Patent application title: Use of Repeat Sequence Protein Polymers in Personal Care Compositions
Inventors:
Manoj Kumar (Fremont, CA, US)
Assignees:
DANISCO US INC.
IPC8 Class: AA61K864FI
USPC Class:
514 23
Class name: Designated organic active ingredient containing (doai) peptide (e.g., protein, etc.) containing doai micro-organism destroying or inhibiting
Publication date: 2013-03-07
Patent application number: 20130059772
Abstract:
The present invention provides personal care compositions, and more
particularly, personal care compositions comprising a bioactively
effective amount of a repeat sequence protein polymer. In some
particularly preferred embodiments, the present invention provides
personal care compositions comprising an effective amount of at least one
fragment of a repeat sequence protein polymer having bioactivity.Claims:
1. A personal care composition comprising an effective amount of a repeat
sequence protein polymer or a fragment thereof, and a physiologically
acceptable carrier or excipient, wherein said repeat sequence protein
polymer provides at least one cosmetic benefit.
2. The personal care composition of claim 1, wherein said cosmetic benefit comprises an increase in skin fibroblast viability.
3. The personal care composition of claim 1, wherein said cosmetic benefit comprises stimulation of collagen synthesis.
4. The personal care composition of claim 1, wherein said cosmetic benefit comprises stimulation of elastin synthesis.
5. The personal care composition of claim 1, wherein said cosmetic benefit comprises inhibition of MMPI activity.
6. The personal care composition of claim 1, wherein said cosmetic benefit comprises inhibition of elastase activity.
7. The composition of claim 1, wherein said repeat sequence protein polymer comprises from about 0.001 weight % to about 10 weight % of the composition.
8. The composition of claim 1, wherein said repeat sequence protein polymer comprises from about 0.01 weight % to about 5 weight % of the composition.
9. The composition of claim 1, wherein said repeat sequence protein polymer comprises from about 0.01 weight % to about 1 weight % of the composition.
10. The composition of claim 1, wherein said fragment is less than 20 amino acids
11. The composition of claim 1, said fragment comprises an amino acid sequence with fewer than 20% of the total number of amino acids in the repeat sequence protein polymer.
12. The composition of claim 1, wherein said fragment comprises a-repeating amino acid sequence with a further biological or chemical function or activity.
13. The composition of claim 1, wherein said fragment comprises at least a portion of the sequence set forth in SEQ ID NOS:25, 26, 27, 28, 30, 31, 32, 34, and 36.
14. The composition of claim 1, wherein said fragment comprises a portion of SEQ ID NO:19.
15-17. (canceled)
18. The composition of claim 1, wherein said personal care composition is a skin care composition selected from moisturizing body washes, body washes, antimicrobial cleansers, skin protective creams, body lotions, facial creams, moisturizing creams, facial cleansing emulsions, surfactant-based facial cleansers, facial exfoliating gels, anti-acne treatments, facial toners, exfoliating creams, facial masks, after shave balms and radioprotectives.
19-21. (canceled)
22. The composition of claim 1, wherein said personal care composition is a makeup composition.
23. The composition of claim 22, wherein said makeup composition is selected from eye gels, high-melting point lipsticks, lipsticks, lip glosses, lip balms, mascaras, eyeliners, pressed powder formulations, and foundations.
24-27. (canceled)
28. A personal care composition comprising an effective amount of a repeat sequence protein polymer or a fragment thereof, with the balance of the composition comprising one or more compounds from the group of carriers, excipients, liposomes, active ingredients, biological or botanical products, humectants, emollients, surfactants, thickening agents, silicone components, organic sunscreens, preservatives, neutralizing agents, perfumes or pigments, and wherein said repeat sequence protein polymer or fragment thereof improves skin tone.
29. A method for making the personal care composition of claim 1, comprising combining an effective amount of a repeat sequence protein polymer fragment or a fragment thereof, with a physiologically acceptable carrier or excipient to obtain a personal care composition.
30. The method of claim 29, further comprising combining a compound from the group of carriers, excipients, liposomes, active ingredients, humectants, emollients, surfactants, thickening agents, silicone components, organic sunscreens, preservatives, neutralizing agents, perfumes or pigments to the composition.
31-36. (canceled)
Description:
RELATED APPLICATIONS
[0001] This application is a Continuation-in-Part of U.S. patent application Ser. No. 10/800,179, filed Mar. 12, 2004, which claims priority under 35 U.S.C. ยง119 of U.S. Provisional Application Ser. No. 60/454,077 filed Mar. 12, 2003.
FIELD OF THE INVENTION
[0002] The present invention provides personal care compositions, and more particularly, personal care compositions comprising a bioactively effective amount of a repeat sequence protein polymer. In some particularly preferred embodiments, the present invention provides personal care compositions comprising an effective amount of at least one fragment of a repeat sequence protein polymer having bioactivity.
BACKGROUND OF THE INVENTION
[0003] Proteins have been widely used as ingredients in personal care products, pigment coating, and in bandages to promote wound healing. Use of silk proteins to perform a variety of functions and to impart desired characteristics to product formulations has been described (See e.g., U.S. Pat. No. 5,747,015; Japanese Patent No. 27186; and Japanese Patent No. 309816). In these disclosures, silk protein hydrolysates were used because of the low solubility of intact silk proteins. For example, for these reasons, proteins have been used to impart some beneficial coating effects such as manageability and strength to hair, to moisturize skin and hair, and to provide film formation to improve the appearance of skin and hair. Proteins have also been used to provide durability properties to many personal care products.
[0004] However, such proteins may not exhibit all desired characteristics when used in personal care products. For example, natural silk proteins may impart durability but may also form tight, hard fibers that are not suitable for film formation. Also, many natural proteins have a low isoelectric point, which reduces the affinity of the protein for the negatively charged skin and hair. Additionally, when more than one protein is needed to impart all desired characteristics to a given formulation, the necessity of using more than one protein may increase the cost and production time for a given personal care product.
[0005] Furthermore, proteins generally have poor solubility due to high molecular weight and hydrophobicity. Commercially available proteins, including structural proteins such as silk and collagen, are typically chemically degraded giving a diverse mixture of molecular weight fragments with variable properties (See e.g., German Patent No. 3139438; and U.S. Pat. No. 6,280,747). However, in most embodiments, hydrolyzed proteins have not been found to be as effective as the intact proteins, as the beneficial effects of the self-assembly, nanofibrilation and coating properties have not realized. As such, these proteins are often modified chemically (See e.g., .U.S. Pat. No. 6,296,860) to enhance solubility for inclusion in personal care products and coating pigments for personal care use (See e.g., U.S. Pat. No. 4,325,741). However, even chemically modified proteins may not have all desired characteristics.
[0006] In addition, in the wound care area, enzymatically digested silk protein has been described as being useful as a healing promoter in wound dressings (See e.g., U.S. Pat. No. 5,382,431). However, digested silk protein is not as effective as water-soluble silk protein (See e.g., WO 04/044172). U.S. Pat. No. 6,175,053 describes the use of dissolved silk proteins as a wound dressing material. However, these preparations required harsh solvents to dissolve the silk protein prior to use.
[0007] Thus, there remains a need in the art for personal care compositions that have desired characteristics without undesirable chemical modification of the proteins. There also remains a need in the art for a method of delivering a protein into a personal care composition so as to effectively deliver the protein in a useable form.
SUMMARY OF THE INVENTION
[0008] The present invention provides to personal care compositions, and more particularly, personal care compositions comprising a bioactively effective amount of a repeat sequence protein polymer. In some particularly preferred embodiments, the present invention provides personal care compositions comprising an effective amount of at least one fragment of a repeat sequence protein polymer having bioactivity.
[0009] In some embodiments, the present invention is directed to personal care compositions comprising an effective amount of at least one repeat sequence protein polymer. In one embodiment, the repeat sequence polymer comprises a repeating amino acid sequence unit derived from elastin, collagen, abductin, byssus, flagelliform silk, dragline silk, gluten high molecular weight subunit, titin, fibronectin, leminin, gliadin, glue polypolypeptide, ice nucleating protein, keratin mucin, RNA polymerase II, resalin or a mixture thereof.
[0010] In another embodiment of the of the invention, the repeat sequence protein polymer (RSPP) formula comprises:
Ty[(An)x(B)b(A'n')x'(B')b'(A''V'').su- b.x'']iTy'
wherein: T and T' each comprise an amino acid sequence of from about 1 to about 100 amino acids, wherein the amino acid sequence of T' is the same as or different from the amino acid sequence of T; y and y' are each an integer from 0 to 1, wherein the integer of y' is the same as or different from the integer of y; A, A' and A'' are each individual repeating amino acid sequence units comprising from about 3 to about 30 amino acids, wherein the amino acid sequence of A' and the amino acid sequence of A'' are the same as or different from the amino acid sequence of A; n, n', and n'' are each integers of at least 2 and not more than 250; x, x' and x'' are each 0 or an integer of at least 1, wherein each integer varies to provide for at least 30 amino acids in the A', A' and A'' individual amino acid sequence repeating units, and wherein the integer of x' and the integer of x'' are the same as or different from the integer of x; B and B' each comprise an amino acid sequence of from about 4 to about 50 amino acids, wherein the amino sequence of B' is the same as or different from the amino acid sequence of B; b and b' are each an integer from 0 to 3, wherein the integer of b' is the same as or different from the integer of b; and i is an integer from 1 to 100.
[0011] In alternative embodiments, the present invention provides bioactive protein polymers, which show efficacy in cell viability improvement, elastin biosynthesis, collagen biosynthesis, and inhibition of skin metelloprotease MMP1 activity.
[0012] The present invention further provides personal care compositions comprising an effective amount of a repeat sequence protein polymer (RSPP) or a fragment thereof, and a physiologically acceptable carrier or excipient, wherein the repeat sequence protein polymer provides at least one cosmetic benefit.
[0013] In some most preferred embodiments, the present invention provides personal care compositions comprising an effective amount of a repeat sequence protein polymer or a fragment thereof, and a physiologically acceptable carrier or excipient, wherein the repeat sequence protein polymer provides improved skin tone.
[0014] In some particularly preferred embodiments, the present invention provides personal care compositions comprising an effective amount of a repeat sequence protein polymer or a fragment thereof, and a physiologically acceptable carrier or excipient, wherein the repeat sequence protein polymer increases skin fibroblast viability.
[0015] In alternative preferred embodiments, the present invention provides personal care compositions comprising an effective amount of a repeat sequence protein polymer or a fragment thereof, and a physiologically acceptable carrier or excipient, wherein the repeat sequence protein polymer stimulates synthesis of collagen.
[0016] In further preferred embodiments, the present invention provides personal care compositions comprising an effective amount of a repeat sequence protein polymer or a fragment thereof, and a physiologically acceptable carrier or excipient, wherein the repeat sequence protein polymer stimulates synthesis of elastin.
[0017] In additional preferred embodiments, the present invention provides personal care compositions comprising an effective amount of a repeat sequence protein polymer or a fragment thereof, and a physiologically acceptable carrier or excipient, wherein the repeat sequence protein polymer inhibits MMP1 activity.
[0018] In still further preferred embodiments, the present invention provides personal care compositions comprising an effective amount of a repeat sequence protein polymer or a fragment thereof, and a physiologically acceptable carrier or excipient, wherein the repeat sequence protein polymer inhibits elastase activity.
[0019] In some embodiments, the repeat sequence protein polymer comprises from about 0.001 weight % to about 10 weight % of the composition, while in other embodiments, the repeat sequence protein polymer comprises from about 0.01 weight % to about 5 weight % of the composition, and in still further embodiments, the repeat sequence protein polymer comprises from about 0.01 weight % to about 1 weight % of the composition.
[0020] In some embodiments of the present invention, the RSPP fragment is less than 20 amino acids, while in further embodiments, the fragment comprises an amino acid sequence with fewer than 20% of the total number of amino acids in the repeat sequence protein polymer. In additional embodiments, the RSPP fragment comprises a repeating amino acid sequence with a further biological or chemical function or activity. In some particularly preferred embodiments, the fragment comprises at least a portion of the sequence set forth in SEQ ID NOS:25, 26, 27, 28, 30, 31, 32, 34, and 36. In further more particularly preferred embodiments, the fragment comprises a portion of SEQ ID NO:19.
[0021] In some embodiments, the RSPP fragment comprises from about 0.001 weight % to about 10 weight % of the composition, while in other embodiments, the RSPP fragment comprises from about 0.01 weight % to about 5 weight % of the composition, and in still other embodiments, the fragment comprises from about 0.01 weight % to about 1 weight % of the composition.
[0022] In additional embodiments, the personal care compositions of the present invention are skin care compositions selected from moisturizing body washes, body washes, antimicrobial cleansers, skin protective creams, body lotions, facial creams, moisturizing creams, facial cleansing emulsions, surfactant-based facial cleansers, facial exfoliating gels, anti-acne treatments, facial toners, exfoliating creams, facial masks, after shave balms and radioprotectives.
[0023] In some embodiments, the personal care composition are skin care compositions comprising topically applied over-the-counter compositions, anti-fungal treatments, anti-acne treatments, skin protectants, and antiperspirants.
[0024] In further embodiments, the personal care compositions are makeup compositions. In some preferred embodiments, the makeup compositions are selected from eye gels, high-melting point lipsticks, lipsticks, lip glosses, lip balms, mascaras, eyeliners, pressed powder formulations, and foundations. In some particularly preferred embodiments, the skin care compositions are radioprotective. In some more particularly preferred embodiments, the radioprotective is a sunscreen selected from non-water-resistant sunscreens, very water-resistant sunscreens, and water-in-silicone sunscreens.
[0025] In further embodiments, the makeup compositions of the present invention comprise at least one pigment. In some preferred embodiments, the makeup compositions comprising at least one pigment are mascaras selected from non-waterproof mascaras, waterproof mascaras, volumizing mascaras, lengthening mascaras, curling mascaras, anhydrous waterproof mascaras, water-based mascaras, and eyelash or eyebrow treatment.
[0026] In further embodiments, the makeup compositions are pressed powder formulations selected from loose powders, blushes, eye shadows, and bronzing powders. In still additional embodiments, the makeup compositions are foundations selected from water-in-oil foundations, water-in-silicone foundations, oil-in-water foundations, anhydrous makeup sticks, and cream-to-powder foundations.
[0027] In some particularly preferred embodiments, the present invention provides personal care compositions comprising an effective amount of a repeat sequence protein polymer or a fragment thereof, with the balance of the composition comprising one or more compounds from the group of carriers, excipients, liposomes, active ingredients, biological or botanical products, humectants, emollients, surfactants, thickening agents, silicone components, organic sunscreens, preservatives, neutralizing agents, perfumes or pigments, and wherein the repeat sequence protein polymer or fragment thereof improves or enhances skin tone.
[0028] The present invention also provides methods for making personal care compositions comprising combining an effective amount of a repeat sequence protein polymer fragment or a fragment thereof, with a physiologically acceptable carrier or excipient to provide a personal care composition.
[0029] In some embodiments, these methods further comprise combining a compound from the group of carriers, excipients, liposomes, active ingredients, humectants, emollients, surfactants, thickening agents, silicone components, organic sunscreens, preservatives, neutralizing agents, perfumes or pigments to the composition.
[0030] The present invention also provides methods for protecting skin from environmental damage comprising the steps of: providing skin to be exposed to environmental damage; providing a composition comprising a repeat sequence protein polymer or at least one fragment of a repeat sequence protein polymer; and applying the composition to the skin. In some embodiments, the environmental damage comprises damage due to exposure to radiation. In further embodiments, the radiation comprises ultraviolet light. In still other embodiments, the environmental damage comprises damage due to exposure to chemicals or free radicals. In some particularly preferred embodiments, the repeat sequence protein polymer comprises SELP47K. In additional particularly preferred embodiment, the repeat sequence protein polymer comprises SEQ ID NO:19.
[0031] In accordance with another embodiment of the present invention, the personal care composition comprises a hair care composition, a skin care composition, a nail care composition, a cosmetic composition, an over-the-counter pharmaceutical composition, or a combination thereof.
[0032] The personal care compositions comprising an effective amount of a repeat sequence protein polymer are advantageous in providing personal care products that have desired characteristic(s) without chemical modifications of the protein. These characteristics include but are not limited to, transparent film formation, hydrogel formation, better efficacy and binding to skin, hair, nail and oral surfaces, desired level of hydrophobicity with water solubility, imparting luster, softness, moisture retainment, mechanical properties (such as tensile properties, viscoelastic behavior, glass transition temperature, cloud temperature, and decomposition temperature). Still other advantages of the present invention will become apparent to those skilled in the art from the following detailed description where alternative exemplary embodiments of this invention are shown and described. As will be realized, the invention is capable of other different, obvious aspects and embodiments, all without departing from the invention. Accordingly, the drawings and descriptions should be regarded as illustrative in nature and not as restrictive.
DESCRIPTION OF THE FIGURES
[0033] FIG. 1 is a chart illustrating stress strain curves of a repeat protein polymer in accordance with an embodiment of the present invention.
[0034] FIG. 2 illustrate AFM image of SELP 47-K film showing self-assembly into nanofilaments.
[0035] FIG. 3 illustrates SEM image of SELP 47-K film showing self-assembly into nanofilaments.
[0036] FIG. 4 provides the sequences for SEQ ID NOS:1-31.
DESCRIPTION OF THE INVENTION
[0037] The present invention provides personal care compositions, and more particularly, personal care compositions comprising a bioactively effective amount of a repeat sequence protein polymer. In particularly preferred embodiments, the present invention provides personal care compositions comprising an effective amount of at least one fragment of a repeat sequence protein polymer having bioactivity.
DEFINITIONS
[0038] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991) provide those of skill in the art with a general dictionaries of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular "a", "an" and "the" includes the plural reference unless the context clearly indicates otherwise. To facilitate understanding of the invention, a number of terms are defined below.
[0039] As used herein, the term "repeat sequence protein polymer" (RSPP) refers to a polymer comprising repeating amino acid sequence units, wherein the repeating units are derived from a natural or synthetic protein. For example, in some embodiments, the repeating sequence units are derived from natural structure supporting materials such as silk, elastin, and collagen. In alternative embodiments, the repeating sequence units are derived from synthetic structures. In some particularly preferred embodiments, the RSPP is SELP47K, while in other preferred embodiments, the RSPP is a variant of SELP47K, while in still further preferred embodiments, the RSPP is a portion of SELP47K (e.g., monomeric SELP47K).
[0040] As used herein, the term "multimer" refers to a portion of the repeat sequence protein polymer. In preferred embodiments, "multimer" refers to a portion of the repeat sequence protein polymer represented by [(An)x(B)b(A'n')x'(B')b'(A''n'')x- '']i in the formula Ty[(An)x(B)b(A'n')x'(B')b'(A''n''- )x''],T'y', described in greater detail in the Detailed Description of the Invention.
[0041] As used herein, "the spacing between repeating sequence units" refers to the other amino acid sequences represented by B or B' in the above formula. In some embodiments, the copolymers are combinations of silk units and elastin units, which provides silk-elastin copolymers having properties distinctive from polymers having only the same monomeric unit.
[0042] A "variant" of an RSPP (e.g., SELP) as used herein, refers to an amino acid sequence that is altered by one or more amino acids. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "nonconservative" changes (e.g., replacement of a glycine with a tryptophan). Similar minor variations may also include amino acid deletions or insertions, or both. In addition to the teaching herein, guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing bioactivity may be found using computer programs well known in the art, for example, DNASTAR software.
[0043] The term "wild-type" refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene. In contrast, the term "modified" or "mutant" refers to a gene or gene product which displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
[0044] The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide or precursor. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties of the full-length or fragment are retained. The term also encompasses the coding region of a structural gene and the including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences. The sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene which are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences which are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene. The 3' flanking region may contain sequences which direct the termination of transcription, post-transcriptional cleavage and polyadenylation.
[0045] As used herein, the term "amino acid sequence" refers to an amino acid sequence of a naturally occurring protein molecule. "Amino acid sequence" and like terms, such as "polypeptide" and "protein" are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
[0046] As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus encodes the amino acid sequence.
[0047] DNA molecules are said to have "5' ends" and "3' ends" because mononucleotides are reacted to make oligonucleotides or polynucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotides or polynucleotide, referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide or polynucleotide, also may be said to have 5' and 3' ends. In either a linear or circular DNA molecule, discrete elements are referred to as being "upstream" or 5' of the "downstream" or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA strand. The promoter and enhancer elements which direct transcription of a linked gene are generally located 5' or upstream of the coding region However, enhancer elements can exert their effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.
[0048] As used herein, the terms "an oligonucleotide having a nucleotide sequence encoding a gene" and "polynucleotide having a nucleotide sequence encoding a gene," refer to a nucleic acid sequence comprising the coding region of a gene (i.e., the nucleic acid sequence which encodes a gene product). The coding region may be present in either a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide or polynucleotide may be single-stranded (i.e., the sense strand) or double-stranded. Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.
[0049] As used herein, the term "regulatory element" refers to a genetic element which controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc. (defined infra).
[0050] As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence "A-G-T," is complementary to the sequence "T-C-A." Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.
[0051] As used herein, the term "homology" refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid is referred to using the functional term "substantially homologous." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.
[0052] The art knows well that numerous equivalent conditions may be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions. In addition, the art knows conditions which promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).
[0053] When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term "substantially homologous" refers to any probe which can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.
[0054] When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to any probe which can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.
[0055] As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acids.
[0056] As used herein, the term "Tm" is used in reference to the "melting temperature." The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the Tm of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (See e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization
[1985]). Other references include more sophisticated computations which take structural as well as sequence characteristics into account for the calculation of Tm.
[0057] As used herein the term "stringency" is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. With "high stringency" conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences. Thus, conditions of "weak" or "low" stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less.
[0058] "Amplification" is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of "target" specificity. Target sequences are "targets" in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out.
[0059] As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
[0060] As used herein, the term "recombinant DNA molecule" as used herein refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques.
[0061] The terms "in operable combination," "in operable order," and "operably linked" as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
[0062] As used herein, the term "isolated" when used in relation to a nucleic acid, as in "an isolated oligonucleotide" or "isolated polynucleotide" refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is such present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids as nucleic acids such as DNA and RNA found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNA s which encode a multitude of proteins. The isolated nucleic acid, oligonucleotide, or polynucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid, oligonucleotide or polynucleotide is to be utilized to express a protein, the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide or polynucleotide may single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be double-stranded).
[0063] As used herein, the term "purified" or "to purify" refers to the removal of contaminants from a sample.
[0064] A compound is said to be "in a form suitable for administration" when the compound may be administered to a human or other animal by any desired route (e.g., topical, oral, etc.). A "composition comprising a given polynucleotide sequence" as used herein refers broadly to any composition containing the given polynucleotide sequence. The composition may comprise an aqueous solution. Compositions comprising polynucleotide sequences encoding SELP47K (e.g., SEQ ID NO:19), variants (e.g., the variants listed in Table 2), or fragments (e.g., monomeric SELP47K) thereof find use in various embodiments of the present invention.
[0065] As used herein, "monodispersed" polymers refers to polymers that have a single defined molecular weight.
[0066] As used herein, "polydispersed" polymers refers to polymers that have been subjected to proteolysis or other means of subdivision, and have a distribution of molecular weights.
[0067] As used herein, the term "personal care composition" refers to a product for application to the skin, hair, nails, oral cavity and related membranes for the purposes of improving, cleaning, beautifying, treating, and/or caring for these surfaces and membranes.
[0068] As used herein, "cosmetic composition" refers to compositions that find use in the cosmetics. The Food Drug and Cosmetic Act (FD&C Act) definition is used herein. Thus, cosmetics are defined by their intended use, as articles intended to be rubbed, poured, sprinkled, or sprayed on, introduced into, or otherwise applied to the human body for cleansing, beautifying, promoting attractiveness, or altering appearance. These compositions provide non-therapeutic benefits and are not regulated as pharmaceuticals. However, in some situations, cosmetic compositions are incorporated into pharmaceutical compositions to provide cosmetic benefits (e.g., products that treat skin or hair diseases, but also contain cosmetic compositions for their coloring or other benefits). Also, it is intended that the present invention encompass the use of cosmetics on animals other than humans.
[0069] As used herein, the terms "pharmaceutical compositions" and "therapeutic compositions" refer to compositions such as drugs that provide medical benefits, rather than solely cosmetic benefits. In the United States, pharmaceutical and therapeutic compositions are approved by the Food and Drug Administration for treatment and/or prevention of particular conditions.
[0070] As used herein, the term "drug" is defined as it is in the FD&C Act definition. Thus, drugs are defined as articles intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease, and articles (other than food) intended to affect the structure or any function of the body of man or other animals.
[0071] As used herein, the term "cosmetic benefit" refers to a desired cosmetic change that results from the administration of a personal care composition. Cosmetic benefits include but are not limited to improvements in the condition of skin, hair, nails, and the oral cavity. In preferred embodiments, at least one cosmetic benefit is provided by the skin care, hair care, nail care, and makeup compositions of the present invention.
[0072] As used herein, "skin care composition" refers to compositions that are applied to skin in order to provide beneficial properties, including but not limited to wrinkle minimizing, wrinkle removal, decoloring, coloring, skin softening, skin smoothing, dipilation, cleansing, etc. In some particularly preferred embodiments, the present invention provides skin care compositions that improve skin tone. In these embodiments, the improvement comprises lessening of wrinkles, smoothing skin texture, modifying skin coloration, and other desired cosmetic benefits.
[0073] As used herein, "hair care composition" refers to compositions that are applied to hair to provide beneficial properties such as thickening, thinning, coloring, decoloring, cleansing, conditioning, softening, shaping, etc.
[0074] As used herein "nail care composition" refers to compositions that are applied to the nails to provide beneficial properties such as harder and stronger nails, nail color, etc.
[0075] As used herein, "makeup compositions" refer to cosmetic preparations that are used to beautify, caring for, maintaining, or augment the appearance of a human or other animal "Makeup compositions" include, but are not limited to color cosmetics, such as mascaras, lipsticks, lip liners, eye shadows, eye-liners, rouges, face powders, foundations, blushes, and nail polish.
[0076] As used herein, the term "an effective amount" refers to the amount of repeat sequence protein polymer, which is added to a personal care composition to provide the composition with a desired characteristic(s).
[0077] As used herein, the term "bioactivity" refers to a cause and effect relationship between a composition and a biological system. Thus, the term is used as by those skilled in the art of biotechnology and biological sciences as the phrase that describes a cause and effect relationship between a molecular composition and living biological matter (e.g., tissue, cells, etc.).
[0078] As used herein as a noun, the term "bioactive" refers a composition that exhibits bioactivity upon administration to living biological matter (e.g., tissue, cells, etc.). The term is used synonymously with "bioactive compound."
[0079] As used herein, the term "dispersed phase" is used as by those of skill in the art of emulsion technology as the phase that exists as small particles or droplets suspended in and surrounded by a continuous phase. The dispersed phase is also known as the "internal" or "discontinuous" phase.
[0080] As used herein, "penetration enhancers" refer to compositions that facilitate penetration through the upper stratum corneum barrier to the deeper skin layers. Examples of penetration enhancers include, but are not limited to, propylene glycol, azone, ethoxydiglycol, dimethyl isosorbide, urea, ethanol, dimethyl sulfoxide, micoroemulsions, liposomes, and nanoemulsions.
[0081] As used herein, the terms "emulsifier" and "surfactant" refer to compounds that disperse and suspend the dispersed phase within the continuous phase of a material. Surfactants find particular use in products intended for skin and/or hair cleansing. In particular embodiments, the term "surfactant(s)" is used in reference to surface-active agents, whether used as emulsifiers or for other surfactant purposes such as skin cleansing.
DETAILED DESCRIPTION OF THE INVENTION
[0082] The present invention provides personal care compositions, and more particularly, personal care compositions comprising a bioactively effective amount of a repeat sequence protein polymer. In some particularly preferred embodiments, the present invention provides personal care compositions comprising an effective amount of at least one fragment of a repeat sequence protein polymer having bioactivity.
[0083] Those skilled in the art appreciate the various naturally occurring proteins containing repeating sequence units which can be used for producing the repeat sequence protein polymers of the present invention, any of which may be employed herein. More specifically, there are more than six hundred repeating amino acid sequence units known to exist in biological systems. For example, well known proteins containing repeating amino acid sequence units include abductin, elastin, byssus, flagelliform silk, dragline silk, gluten high molecular weight (HMW) subunit, titin, fibronectin, leminin, and collagen.
[0084] Individual repeating amino acid sequence units of particular interest include units found in silk-, elastin-, collagen-, abductin-, byssus-, gluten-, titin-, extensin-, and fibronectin-like proteins. Silk-like proteins comprise a repeating sequence unit SGAGAG (SEQ ID NO:1). This repeating sequence unit is found in naturally occurring silk fibroin protein, which can be represented as GAGAG(SGAGAG)8SGAAGY (SEQ ID NO:2). Elastin-like proteins comprise a base repeating sequence unit of GVGVP (SEQ ID NO:3). This repeating sequence unit may be found in naturally occurring elastin. Collagen-like proteins comprise a repeating sequence unit of G-X-X1, wherein X comprises any amino acid, often alanine or proline; and X1 comprises any amino acid, often proline or hydroxy-proline (SEQ ID NO:20). In one embodiment, collagen-like protein comprises SEQ ID NO:29. Abductin-like proteins comprise a base repeating sequence unit of GGFGGMGGGX, wherein X comprises any amino acid (SEQ ID NO:4). Byssus-like proteins comprise a repeating sequence unit of GPGGG (SEQ ID NO:5). Gluten-like proteins of the high molecular weight subunit comprise repeating sequence units of PGQGQQ (SEQ ID NO:6), GYYPTSPQQ (SEQ ID NO:7), and GQQ (SEQ ID NO:8). Titin-like proteins comprise repeating sequence units of PPAKVPEVPKKPVPEEKVPVPVPKKPEA (SEQ ID NO:9), which proteins may be found in the heart, psoas, and soleus muscle. Extensin-like proteins comprise repeating sequence units of SPPPPSPKYVYK (SEQ ID NO:10). Fibronectin-like proteins comprise repeating sequence units of RGDS (SEQ ID NO:11). Additional repeating sequence units are found, for example, in gliadin, glue polypolypeptide, ice nucleating protein, keratin, mucin, RNA polymerase II, and resilin. Gliadin comprises a repeating sequence unit of PQQPY (SEQ ID NO:12). The glue polypeptide comprises a repeating sequence unit of PTTTK (SEQ ID NO:13). The ice nucleating protein comprises a repeating sequence unit of AGYGSTGT (SEQ ID NO:14). Keratin comprises repeating sequence units of YGGSSGGG (SEQ ID NO:15) or FGGGS (SEQ ID NO:16). Mucin comprises a repeating sequence unit of TTTPDV (SEQ ID NO:17). RNA polymerase II comprises a repeating sequence unit of YSPTSPS (SEQ ID NO:18).
[0085] In addition to repeating units derived from naturally occurring proteins, synthetic repeating amino acid sequences units may be utilized. In a particular embodiment, the repeat sequence protein polymer has the formula:
Ty[(An)x(B)b(A'n')x'(B'')b'(A''n- '')x''iT'y'
[0086] wherein:
[0087] T and T' each comprise an amino acid sequence of from about 1 to about 100 amino acids, specifically an amino acid sequence of from about 1 to about 60 amino acids, and more specifically an amino acid sequence with fewer than 20% of the total number of amino acids in the repeat sequence protein polymer, wherein the amino acid sequence of T' is the same as or different from the amino acid sequence of T;
[0088] y and y' are each an integer from 0 to 1, wherein the integer of y' is the same as or different from the integer of y;
[0089] A, A' and A'' are each individual repeating sequence units comprising from about 3 to about 30 amino acids, wherein the amino acid sequence of A' and the amino acid sequence of A'' are the same as or different from the amino acid sequence of A;
[0090] n, n', and n'' are each integers of at least 2 and not more than 250;
[0091] x, x' and x'' are each 0 or an integer of at least 1, wherein each integer varies to provide for at least 30 amino acids in the A', A' and A'' individual repeating sequence units, and wherein the integer of x' and the integer of x'' are the same as or different from the integer of x;
[0092] B and B' each comprise an amino acid sequence of from about 4 to about 50 amino acids, wherein the amino sequence of B' is the same as or different from the amino acid sequence of B;
[0093] b and b' are each an integer from 0 to 3, wherein the integer of b' is the same as or different from the integer of b; and
[0094] i is an integer from 1 to 100, specifically from 1 to 50, and more specifically from 1 to 30.
[0095] Additionally, in some embodiments, the repeat sequence protein polymer comprises amino acid sequences that link the repeating A, A', and A'' units or amino acid sequences that link between the individual A, A' or A'' repeating sequence units. In some preferred embodiments, the linking sequences are from about 1 to about 10 amino acids.
[0096] Those skilled in the art appreciate the various methods for producing the repeat sequence protein polymers of the present invention, any of which may be employed herein. For example, the repeat sequence protein polymer may be produced by generally recognized methods of chemical synthesis (See e.g., Andersson et. al., Biopolymers 55:227-50
[2000]); genetic manipulation (See e.g., Cappello, Genetically Engineered Protein Polymers, In Handbook of Biodegradable Polymers, Domb et al. (eds), Harvard Academic Publishers, Amsterdam; pages 387-414), and enzymatic synthesis (See e.g., Wong and Wang, Experientia 47:1123-9
[1991]). Furthermore, in other embodiments, the repeat sequence protein polymers of the present invention are produced using the methods described in U.S. Pat. Nos. 5,243,038 and 6,355,776, the disclosures of which are incorporated by reference herein. In yet additional embodiments, the repeat sequence protein polymers are produced utilizing non-ribosomal peptide synthase (See e.g., Dohren et al., Chem. Rev., 97:2675-2705
[1997]). In some particularly preferred embodiments, the repeat sequence protein polymers are produced on a commercial scale.
[0097] In some embodiments, the repeating amino acid sequence units comprise identical repeating sequence units, while in other embodiments, they comprise different repeating sequence unit combinations, which join together to form a block copolymer or an alternating block copolymer. Additionally, in some embodiments, the individual repeating amino acid sequence units of the repeat sequence protein polymer comprise from about 3 to about 30 amino acids. In other embodiments, the individual repeating units comprise from about 3 to about 8 amino acids. Moreover, the same amino acid may appear at least twice in the same repeating sequence unit. Thus, it is not intended that the amino acids be limited as to the number of repeats in each repeating sequence unit.
[0098] Repeat sequence protein polymers utilizing natural and/or synthetic repeating sequence units are produced to provide various desirable characteristics. One skilled in the art appreciates the various desirable characteristics for repeat sequence protein polymers, any of which may be employed herein. The characteristics may include, for example, moisturizing properties, adhesion, contraction, entrapment, high glass transition temperature for hardness or strength, a high cloud temperature for heat sensitive applications, and/or a high isoelectric point to increase the affinity of the protein to hair, skin, and nails, etc. Self-assembly and nanofilament formation properties find use in skin anti-wrinkle and fine line filling applications. Additionally, in some embodiments, a protein having a particular molecular weight is chosen in order to increase or decrease water solubility and/or other properties as desired.
[0099] Furthermore, repeat sequence protein polymers are advantageous in providing personal care products when modified with desired chemical agents. RSPPs provide amino, hydroxyl and/or carboxyl functional groups that can be covalently reacted, conjugated, composited or ionically bonded with various personal care chemical and formulating functional ingredients. In various embodiments, these include: UV absorbers (e.g., octyl methoxycinnamate, benzophenone-3, titanium dioxide, and octyl salicylate); film-forming agents (e.g., VP/Eicosene copolymer); cosmeceutical agents (e.g., peptides and proteins, alpha hydroxy acids, and retinol and retinoic acid derivatives); antioxidants (e.g., tocopherol and derivatives thereof and ascorbic acid and derivatives thereof); vitamins (e.g., B, D, K and their derivatives); antiperspirant actives (e.g., aluminum hydroxide and zirconium hydroxide); depilating agents (e.g., thioglycolate salts); anti-acne agents (e.g., salicylic acid and benzoyl peroxide); abrasives and exfoliants (e.g., silicates, pumice, and polyethylene); and extracts of plant, fruit, vegetable and/or marine sources.
[0100] It will also be understood by those having skill in the art that in some embodiments, the synthetic repeat sequence protein polymers are produced to have a combination of desirable characteristics. For example, in some embodiments, a copolymer comprising silk repeating sequence units and elastin repeating sequence units is synthesized to impart durability due to the silk repeating sequence units and to impart flexibility due to the elastin repeating sequence units. In additional embodiments, the silk-elastin polymer exhibits other desirable properties such as good clear film and hydrogel formation, which the individual monomeric units may not exhibit. In further embodiments, the silk elastin copolymer is hydrophilic and water-soluble. In yet additional embodiments, the silk elastin copolymer exhibits a high cloud temperature which is desirable in heat sensitive applications. In yet further embodiments, the silk elastin copolymer has a high isoelectric point, which in some embodiments, makes the copolymer more substantive to skin and hair. In additional embodiments, the silk elastin copolymer further exhibits self-assembly into fibers and films, which is desirable in some applications.
[0101] The desired properties of the repeating sequence units are designed to produce the desired characteristics by choosing the particular repeating amino acid sequence units, the number of repeating sequence units in each multimer, the spacing between the repeating sequence units, and the number of repeats of the multimer.
[0102] It will be further understood by those having skill in the art that the repeat sequence protein polymers of the present invention may be monodispersed or polydispersed, as desired.
[0103] In accordance with some embodiments of the present invention, a silk-elastin polymer SELP47K (SEQ ID NO:19) finds use as a repeat sequence protein polymer of the present invention. The SELP47K is a homoblock protein polymer that consists exclusively of silk-like crystalline blocks and elastin-like flexible blocks. SELP47K is more linear than many proteins because it has a beta sheet two-dimensional structure rather than an alpha helix three-dimensional structure. SELP47K exhibits the ability to self-assemble by cross-linking of beta sheets into fibers. SELP47K is 70% proline, valine, and alanine, and has hydrophobic characteristics. Additionally, SELP47K has a high lysine ratio. In additional embodiments, the repeat sequence protein polymer comprises SELP 47-E13 (SEQ ID NO:25), SELP 47R-3 (SEQ ID NO:26), SELP 47K-3 (SEQ ID NO:27), SELP 47 E-3 (SEQ ID NO:28), SELP 67K (SEQ ID NO:30), and/or SELP 58 (SEQ ID NO:31), SELP47K-P4 (SEQ ID NO:32), DCP6 (SEQ ID NO:34).
[0104] In some preferred embodiments, once a suitable repeat sequence protein polymer is been synthesized and purified, an effective amount is added to personal care composition(s) that find use in personal care products. Personal care products can be classified/described as cosmetic, over-the-counter ("OTC") compounds that find use in personal care applications (e.g., cosmetics, skin care, oral care, hair care, nail care). In some embodiments, the repeat sequence protein polymer is added to a personal care composition such as a hair care composition, a skin care composition, a nail care composition, a cosmetic composition, or any combinations thereof.
[0105] In some embodiments, the hair care composition is in a form selected from the group consisting of shampoos, conditioners, anti-dandruff treatments, styling aids, styling conditioners, hair repair or treatment sera, lotions, creams, pomades, and chemical treatments. In other embodiments, the styling aids are selected from the group consisting of sprays, mousses, rinses, gels, foams, and combinations thereof. In further embodiments, the chemical treatments are selected from the group consisting of permanent waves, relaxers, and permanents, semi-permanents, temporary color treatments and combinations thereof. In further embodiments, the skin care composition is in a form selected from the group consisting of body washes, moisturizing body washes, deodorant body washes, antimicrobial cleansers, skin protecting treatments, body lotions, facial creams, moisturizing creams, facial cleansing emulsions, surfactant-based facial cleansers, facial exfoliating gels, facial toners, exfoliating creams, facial masks, after shave lotions, balms, and/or radioprotective compositions (e.g., sunscreens).
[0106] In other embodiments, the cosmetic composition is in a form selected from the group consisting of eye gels, eye shadows, high-melting point lipsticks, lipsticks, lip glosses, lip balms, mascara, brow liners, eyeliners, pressed powder formulations, foundations, protein coated pigments, and combinations thereof. In further embodiments, the cosmetic compositions comprise makeup compositions. In yet another embodiment, the nail care composition is in a form selected from the group consisting of nail enamel, cuticle treatment, nail polish, nail treatment, and polish remover. In yet another embodiment, the oral care composition is in a form selected from the group consisting of toothpaste, mouth rinse, breath freshener, whitening treatment, and inert carrier substrates. In yet another embodiment, the over-the-counter composition comprises radioprotectives (e.g., sunscreens), anti-acne, antiperspirants, skin protectants, anti-dandruff products, anti-fungal, hemorrhoidal ointments, and toothpaste.
[0107] In further embodiments, the personal care composition is in the form of an emulsified vehicle, such as a nutrient cream or lotion, a stabilized gel or dispersioning system, such as skin softener, a nutrient emulsion, a nutrient cream, a massage cream, a treatment serum, a liposomal delivery system, a topical facial pack or mask, a surfactant-based cleansing system such as a shampoo or body wash, an aerosolized or sprayed dispersion or emulsion, a hair or skin conditioner, styling aid, or a pigmented product such as makeup in liquid, cream, solid, anhydrous or pencil form.
[0108] In some preferred embodiments, the repeat sequence protein polymer SELP47K finds use in personal care compositions. Specifically, SELP47K finds used in hair care products, as it is contemplated to provide such benefits as building body and/or volume, repair of damaged hair, and/or protection of hair from chemical damage. In additional embodiments, SELP47K has applications in skin care products, as it is contemplated to provide various benefits such as tightening or firming of the skin, moisturization, improved skin tone, oil absorption, and/or improvement in the appearance of fine lines and wrinkles. It is further contemplated that SELP47K will find use in various make-up products, in particular to provide eyelash flexibility, volume, length, and strength.
[0109] In some embodiments, the repeat sequence protein polymer comprises from about 0.001% to about 10% by weight of the personal care composition. In other embodiments, the repeat sequence protein polymer comprises from about 0.01% to about 5% by weight of the personal care composition. In yet other embodiments, the repeat sequence protein polymer comprises from about 0.01% to about 1% by weight of the personal care composition.
[0110] In some embodiments, the personal care composition comprising an effective amount of a repeat sequence protein polymer, as set forth herein, comprises additional components. For example, in some embodiments, the personal care compositions of the present invention comprise liposomes, wherein the liposomes comprise components such as water and one or more ingredients capable of forming lipid bilayer vesicles that can hold one or more functional or active ingredient(s). Non-limiting examples of ingredients capable of forming lipid bilayer vesicles include: phospholipids, hydrogenated phosphatidylcholine, lecithin, cholesterol and sphingolipids. Non-limiting examples of functional or active ingredients that can be delivered from liposomes include: vitamins and their derivatives, antioxidants, proteins and peptides, keratolytic agents, bioflavinoids, terpenoids, phytochemicals, and extracts of plant, marine or fermented origin. In some embodiments, liposomes include, without limitation: a) lipoid liposome 0003 (composed of water and lecithin and glycerin); b) lipoid liposome 0300 (composed of water and phosphatidylcholine), c) lipoid liposome 0111 (composed of water, ginkgo balboa leaf extract, denatured alcohol, hydrogenated lecithin and cholesterol) d) anti-irritant liposomes (composed of water, cola acuminata seed extract, bisabolol and phospholipids), e) vitamin C and E liposomes (composed of water, phospholipids, tocopheryl acetate and ascorbyl palmitate), f) firming liposomes (composed of water, butylene glycol, pyrus malus (apple) fruit extract, phospholipids, tocopheryl acetate and carbomer) and g) moisturizing liposomes (composed of water, sodium PCA, tocopheryl acetate, xanthan gum, arginine, lysine, glycine and proline).
[0111] In other embodiments, the personal care composition of the present invention further comprise at least one active ingredient. There are numerous active ingredients known to those of skill in the art that find use in the personal care compositions of the present invention. Indeed, it is contemplated that any suitable active ingredient or combination of suitable active ingredients will find use in the present invention (See e.g., McCutcheon's Functional Materials, North American and International Editions, published by MC Publishing Co.
[2003]). For example, in some embodiments, the personal care compositions herein comprise a skin care active ingredient at a level from about 0.0001% to about 20%, by weight of the composition. In another embodiment, the personal care compositions comprise a skin care active ingredient from about 0.001% to about 5%, by weight of the composition. In yet another embodiment, the personal care compositions comprise a skin care active ingredient from about 0.01% to about 2%, by weight of the composition.
[0112] Suitable skin care active ingredients include, but are not limited to, antioxidants (e.g., tocopheryl and ascorbyl derivatives); bioflavinoids, terpenoids, synthetics of biolflavinoids and terpenoids and the like; vitamins and vitamin derivatives; hydroxyl- and polyhydroxy acids and their derivatives (e.g., AHAs and BHAs and their reaction products); peptides and polypeptides and their derivatives (e.g., glycopeptides and lipophilized peptides, heat shock proteins and cytokines); enzymes and enzymes inhibitors and their derivatives (e.g., proteases, MMP inhibitors, catalases, CoEnzyme Q10, glucose oxidase and superoxide dismutase (SOD)); amino acids and their derivatives; bacterial, fungal and yeast fermentation products and their derivatives (e.g., mushrooms, algae and seaweed and their derivatives); phytosterols and plant and plant part extracts; phospholipids and their derivatives; anti-dandruff agents (e.g., zinc pyrithione); and sunscreen agents (e.g., ethylhexyl methoxycinnamate, avobenzone, and phenyl benzimidazole sulfonic acid). Delivery systems comprising the active ingredients are also provided herein.
[0113] In some embodiments, the skin care active ingredient is selected from the group consisting of a Vitamin B3 component, panthenol, Vitamin E, Vitamin E acetate, retinoid, retinol, retinyl, propionate, retinyl palmitate, retinoic acid, Vitamin C, theobromine, alpha-hydroxyacid, farnesol, phytrantriol, salicylic acid, palmityl peptapeptide-3 and mixtures thereof. In other embodiments, the Vitamin B3 compound is niacinamide. In yet other embodiments, the vitamin B3 compound is tocopherol nicotinate. Exemplary derivatives of the foregoing vitamin B3 compounds include nicotinic acid esters (e.g., non-vasodilating esters of nicotinic acid, nicotinyl amino acids, nicotinyl alcohol esters of carboxylic acids, and nicotinic acid N-oxide and niacinamide N-oxide). Suitable esters of nicotinic acid include nicotinic acid esters of C1-C22, specifically C1-C16, more specifically C1-C6 alcohols. The alcohols are suitably straight-chain or branched chain, cyclic or acyclic, saturated or unsaturated (including aromatic), and substituted or unsubstituted. The esters are specifically non-vasodilating. Non-vasodilating esters of nicotinic acid include tocopherol nicotinate and inositol hexanicotinate. Although these compounds are well known to those in the art, a more complete description of vitamin B3 compounds is provided by WO 98/22085.
[0114] In some embodiments, the retinoid skin care active ingredient is selected from the group consisting of retinol, retinol esters (e.g., C2-C22 alkyl esters of retinol, including retinyl palmitate, retinyl acetate, retinyl proprionate), retinal, and/or retinoic acid (including all-trans retinoic acid and/or 13-cis-retinoic acid). These compounds are well known in the art and are commercially available from a number of sources (e.g., Sigma-Aldrich Chemical Company, and Boehringer Mannheim). Exemplary retinoids include retinol, retinyl palmitate, retinyl acetate, retinyl proprionate, retinal, retinoic propionate, retinoic acid and combinations thereof. In some embodiments, the retinoid is included as a substantially pure material, while in other embodiments, it is an extract obtained by suitable physical and/or chemical isolation from natural (e.g., plant) sources. In some embodiments, the retinoid comprises from about 0.005% to about 2% and more preferably from about 0.01% to about 12% by weight of the personal care composition. In other embodiments, the personal care composition comprises retinol. In some preferred embodiments, the retinol comprises from about 0.01% to about 0.15% by weight of the personal care composition. In yet other embodiments, the personal care composition comprise retinol esters. In some preferred embodiments, the retinol esters comprise from about 0.01% to about 2% by weight of the personal care composition.
[0115] In addition to the active ingredients noted above, in some embodiments, the personal care composition comprise a physiologically acceptable carrier and/or excipient. In some particularly preferred embodiments, the personal care compositions herein comprise a safe and effective amount of at least one dermatologically acceptable carrier suitable for topical application to the skin, nails, mucous membranes, and/or hair within which the essential materials and optional other materials are incorporated to enable the essential materials and optional components to be delivered to the site of application (i.e., the target site) at an appropriate concentration. Thus, the carrier acts as a diluent, dispersant, solvent or the like for the essential components which ensures that they can be applied to and distributed evenly over the selected target site at an appropriate concentration.
[0116] In some embodiments, as indicated above, an effective amount of one or more compounds described herein also includes personal care compositions suitable for application to keratinous materials such as nails and hair, including but not limited to those useful as hair spray compositions, hair styling compositions, hair shampooing and/or conditioning compositions, compositions applied for the purpose of hair growth regulation and compositions applied to the hair and scalp for the purpose of treating seborrhoea, dermatitis and/or dandruff.
[0117] In some embodiments, an effective amount of one or more compounds described herein included in personal care compositions suitable for topical application to the skin, teeth, nails or hair. In some embodiments, these compositions are in the form of creams, lotions, gels, suspensions dispersions, microemulsions, nanodispersions, microspheres, hydrogels, emulsions (e.g., oil-in-water and water-in-oil, as well as multiple emulsions) and multilaminar gels and the like (See e.g., Schlossman et al., The Chemistry and Manufacture of Cosmetics,
[1998]). In some embodiments, these compositions are formulated as aqueous or silicone compositions, while in other embodiments they are formulated as emulsions of one or more oil phases in an aqueous continuous phase, and is still further embodiments, are an aqueous phase in an oil phase.
[0118] The type of carrier utilized in the present invention depends on the type of product form desired for the personal care composition. In some embodiments, the carrier is a solid, while in other embodiments, it is semi-solid or liquid. Suitable carriers include liquids, as well as semi-solids (e.g., creams, lotions, gels, sticks, ointments, pastes, sprays and mousses). In particular, carriers that are lotions, creams or gels have sufficient thickness or yield find use in the present invention, as they prevent the particles from sedimenting. In some embodiments, the carrier itself is inert, while in other embodiments, it possesses dermatological benefits of its own. In some embodiments, the carrier is applied directly to the teeth, skin, nails and/or hair, while in other embodiments, it is applied via a woven or non-woven wipe or cloth. In alternative embodiments, it is provided as a patch, mask, wrap, or another inert substrate. In yet further embodiments, the carrier is aerosolized or otherwise sprayed or pumped onto the skin and/or hair. In preferred embodiments, the carrier is physically and chemically compatible with the essential components described herein, and does not unduly impair stability, efficacy or other use benefits associated with the compositions of the present invention.
[0119] In some embodiments of the present invention, the carrier is selected from the group consisting of water, propylene glycol, ethanol, propanol, glycerol, butylene glycol, and polyethylene glycol, as well as any suitable combination thereof. In further embodiments, the carriers also contain at least one dermatologically acceptable, hydrophilic diluent. Suitable hydrophilic diluents include water, organic hydrophilic diluents (e.g., C2-C10, specifically C2-C6, more specifically, C3-C6 monohydric alcohols) and low molecular weight glycols and polyols (e.g., propylene glycol, polyethylene glycol polypropylene glycol, glycerol, butylene glycol, 1,2,4-butanetriol, sorbitol, 1,2,6-hexametriol, pentylene glycol, hexylene glycol, sorbitol esters, ethoxylated ethers, propoxylated ethers) and combinations thereof. In one embodiment, the diluent is a liquid. In one preferred embodiment, the diluent is water. In another embodiment, the personal care composition comprises at least about 20% of the hydrophilic diluent.
[0120] In some embodiments, suitable carriers also comprise an emulsion comprising a hydrophilic phase, especially an aqueous phase, and a hydrophobic phase (e.g., a lipid, oil or oily material). As well known to those skilled in the art, the hydrophilic phase is dispersed in the hydrophobic phase, or vice versa, to form respectively hydrophilic or hydrophobic dispersed and continuous phases, depending on the composition of ingredients. In some embodiments, the emulsion comprises an oil-in-water emulsion or a water-in-oil emulsion such as a water-in-silicone emulsion (e.g., in a triple or other multi-phase emulsion). In some preferred embodiments, oil-in-water emulsions comprise from about 1% to about 60% or from about 1% to about 30% of the dispersed hydrophobic phase, and from about 1% to about 99% or from about 10% to about 90% of the continuous hydrophilic phase, while in some alternative embodiments, water-in-oil emulsions comprise from about 1% to about 98%, or from about 40% to about 90% of the dispersed hydrophilic phase and from about 1% to about 50% or from about 1% to about 30% of the continuous hydrophobic phase.
[0121] In some embodiments, the carrier also includes one or more components that facilitate penetration through the upper stratum corneum barrier to the deeper skin layers. Examples of penetration enhancers include, but are not limited to, propylene glycol, azone, ethoxydiglycol, dimethyl isosorbide, urea, ethanol, dimethyl sulfoxide, micoroemulsions, liposomes and nanoemulsions. However, it is intended that any suitable penetration enhancer will find use in the present invention.
[0122] In some embodiments, the personal care compositions of the present invention further comprise humectants. In some of these embodiments, the personal care composition comprises from about 0.01% to about 20% by weight of humectant. In other embodiments, the personal care composition comprises from about 0.1% to about 15% by weight of a humectant. In yet other embodiments, the personal care composition comprises from about 0.5% to about 10% by weight of a humectant. Humectants that find use in the present invention include, but are not limited to, compounds selected from polyhydric alcohols, sorbitol, glycerol, urea, betaine, D or DL panthenol, calcium pantothenate, royal jelly, panthetine, pantotheine, panthenyl ethyl ether, pangamic acid, pyridoxin, pantoyl lactose Vitamin B complex, sodium pyrrolidone carboxylic acid, hexane-1,2,6, -triol, guanidine or its derivatives, and mixtures thereof. However, it is intended that any suitable humectant will find use in the present invention.
[0123] Examples of suitable polyhydric alcohol humectants for use herein include polyalkylene glycols and specifically alkylene polyols and their derivatives, including propylene glycol, dipropylene glycol, polypropylene glycol, polyethylene glycol and derivatives thereof, sorbitol, hydroxypropyl sorbitol, erythritol, threitol, pentaerythritol, xylitol, glucitol, mannitol, pentylene glycol, hexylene glycol, butylene glycol (e.g., 1,3-butylene glycol), hexane triol (e.g., 1,2,6-hexanetriol), trimethylol propane, neopentyl glycol, glycerine, ethoxylated glycerine, propane-1,3 diol, propoxylated glycerine and mixtures thereof. The alkoxylated derivatives of any of the above polyhydric alcohols are also suitable for use herein. In some embodiments, polyhydric alcohols of the present invention are selected from glycerine, butylene glycol, propylene glycol, pentylene glycol, hexylene glycol, dipropylene glycol, polyethylene glycol, hexane triol, ethoxylated glycerine and propoxylated glycerine and mixtures thereof.
[0124] Additional suitable humectants useful herein include sodium 2-pyrrolidone-5-carboxylate (NaPCA), guanidine, glycolic acid, glycolate salts (e.g., ammonium and quaternary alkyl ammonium), lactic acid, lactate salts (e.g., ammonium and quaternary alkyl ammonium), aloe vera in any of its variety of forms (e.g., aloe vera gel), hyaluronic acid and derivatives thereof (e.g., salt derivatives such as sodium hyaluronate), lactamide monoethanolamine, acetamide monoethanolamine, urea, betaine, panthenol and derivatives thereof, and mixtures thereof.
[0125] In some embodiments, at least part (up to about 5% by weight of composition) of a humectant is incorporated in the form of an admixture with a particulate cross-linked hydrophobic acrylate or methacrylate copolymer, itself specifically present in an amount of from about 0.1% to about 10%, which can be added either to the aqueous or disperse phase. As known in the art (See e.g., WO96/03964), this copolymer is particularly valuable for reducing shine and controlling oil, while helping to provide effective moisturization benefits.
[0126] In some preferred embodiments, the oil-in-water and oil-in-water-in-oil emulsion embodiments of the present invention comprise from about 0.05% to about 20%, specifically from about 1% to about 15%, more specifically from about 2% to about 10%, and even more specifically from about 2% to about 5% by weight of a dermatologically acceptable emollient. Emollients tend to lubricate the skin, increase the smoothness and suppleness of the skin, prevent or relieve dryness of the skin and/or protect the skin. Emollients are typically water-immiscible, oily or waxy materials and emollients with high molecular weights can confer aesthetic properties to a topical composition. A wide variety of suitable emollients are known and find use herein. As known in the art (See, Sagarin, Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp. 32-43
[1972]; and WO 00/24372), numerous materials find use as emollients. Thus, it is intended that any suitable emollient will find use in the present invention. However, some preferred examples are outlined in further detail below:
[0127] i) Straight and branched chain hydrocarbons having from about 7 to about 40 carbon atoms, such as mineral oils, dodecane, squalane, cholesterol, hydrogenated polyisobutylene, isohexadecane, isoeicosane, isooctahexacontane, isohexapentacontahectane, and the C7-C40 isoparaffins, which are C2-C40 branched hydrocarbons. Suitable branched chain hydrocarbons for use herein are selected from isopentacontaoctactane, petrolatum and mixtures thereof;
[0128] ii) C1-C30 fatty acid esters of C1-C30 carboxylic acids, C12-15 12-15 alkyl benzoates and of C2-C30 dicarboxylic acids, such as isononyl isononanoate, isostearyl neopentanoate, isodecyl octanoate, isodecyl isononanoate, tridecyl isononanoate, myristyl octanoate, octyl pelargonate, octyl isononanoate, myristyl myristate, myristyl neopentanoate, myristyl octanoate, isopropyl myristate, myristyl propionate, isopropyl stearate, isopropyl isostearate, methyl isostearate, behenyl behenate, dioctyl maleate, diisopropyl adipate, and diisopropyl dilinoleate and mixtures thereof;
[0129] iii) C1-C30 mono- and poly-esters of sugars and related materials derived from a sugar or polyol moiety and one or more carboxylic acid moieties. Depending on the constituent acid and sugar, these esters are either liquid or solid form at room temperature. Examples include glucose tetraoleate, the galactose tetraesters of oleic acid, the sorbitol tetraoleate, sucrose tetraoleate, sucrose pentaoleate, sucrose hexaoleate, sucrose heptaoleate, sucrose octaoleate, sorbitol hexaester. Other materials include cottonseed oil or soybean oil fatty acid esters of sucrose. Other examples of such materials are described in WO 96/16636;
[0130] iv) Vegetable oils and hydrogenated vegetable oils. Examples of vegetable oils and hydrogenated vegetable oils include safflower oil, grapeseed oil, coconut oil, cottonseed oil, menhaden oil, palm kernel oil, palm oil, peanut oil, soybean oil, rapeseed oil, linseed oil, rice bran oil, pine oil, nut oil, sesame oil, sunflower seed oil, partially and fully hydrogenated oils from the foregoing sources and mixtures thereof; and
[0131] v) Soluble or colloidally-soluble moisturizing agents. Examples include hyaluaronic acid and, chondroitin sulfate, heparan sulfate, and starch-grafted sodium polyacrylates.
[0132] In further embodiments, personal care compositions of the present invention also contain one or more emulsifiers and/or surfactants, generally to help disperse and suspend the disperse phase within the continuous phase. Surfactants find particular use in products intended for skin and/or hair cleansing. Known and/or conventionally used surfactants (See e.g., WO 00/24372) find use in the personal care compositions of the present invention, provided that the selected agent is chemically and physically compatible with essential components of the composition and provides the desired characteristics. Suitable surfactants include non-silicone derived materials, silicone-derived materials, and mixtures thereof. In some embodiments, the personal care compositions of the present invention comprise from about 0.05% to about 30%, preferably from about 0.5% to 15%, and more preferably from about 1% to 10% by weight of a surfactant or mixture of surfactants. As known to those skilled in the art, the exact surfactant or surfactant mixture chosen will depend upon the pH of the composition, the other components present and the desired final product aesthetics.
[0133] Among the nonionic surfactants that are useful herein are those that can be broadly defined as condensation products of long chain alcohols (e.g., C8-30 alcohols, with sugar or starch polymers, such as glycosides). Other useful nonionic surfactants include the condensation products of alkylene oxides with fatty acids (i.e., alkylene oxide esters of fatty acids). These materials have the general formula RCO(X)nOH wherein R is a C10-30 alkyl group, X is --OCH2CH2-- (i.e., derived from ethylene glycol or oxide) or --OCH2CHCH3-- (i.e., derived from propylene glycol or oxide) and n is an integer from 6 to 200. Other nonionic surfactants are the condensation products of alkylene oxides with 2 moles of fatty acids (i.e., alkylene oxide diesters of fatty acids). These materials have the general formula RCO(X)nOOCR wherein R is a C10-30 alkyl group, X is --OCH2CH2-- (i.e., derived from ethylene glycol or oxide) or --OCH2CHCH3-- (i.e., derived from propylene glycol or oxide) and n is an integer from 6 to 100. For example, an emulsifier for use herein is most specifically a fatty acid ester blend based on a mixture of sorbitan fatty acid ester and sucrose fatty acid ester, more specifically a blend of sorbitan stearate and sucrose cocoate. Even further suitable examples include a mixture of cetearyl alcohols and cetearyl glucosides.
[0134] Hydrophilic surfactants useful herein can alternatively or additionally include any of a wide variety of cationic, anionic, zwitterionic, and amphoteric surfactants such as those known in the art (See, e.g., McCutcheon's, Detergents and Emulsifiers and Detergents, North American
[2003], and International Editions
[1986], published by MC Publishing Co. and Allured Publishing Corporation; and U.S. Pat. Nos. 5,011,681, 4,421,769, and 3,755,560, herein incorporated by reference).
[0135] A variety of anionic surfactants are known in the art (See e.g., U.S. Pat. No. 3,929,678, herein incorporated by reference) also find use in the personal care compositions of the present invention. Examples of anionic surfactants include the alkoyl isethionates (e.g., C12-C30), alkyl and alkyl ether sulfates and salts thereof, alkyl and alkyl ether phosphates and salts thereof, alkyl methyl taurates (e.g., C12-C30), and soaps (e.g., substituted alkylamine and alkali metal salts, such as sodium or potassium salts) of fatty acids.
[0136] Amphoteric and zwitterionic surfactants are find use in some embodiments of the personal care compositions of the present invention. Examples of amphoteric and zwitterionic surfactants which find use in the compositions of the present invention include those that are broadly described as derivatives of aliphatic secondary and tertiary amines in which the aliphatic radical can be straight or branched chain and wherein one of the aliphatic substituents contains from about 8 to about 22 carbon atoms (specifically C8-C18) and one contains an anionic water solubilizing group (e.g., carboxy, sulfonate, sulfate, phosphate, or phosphonate). Examples include alkyl imino acetates and iminodialkanoates and aminoalkanoates, imidazolinium and ammonium derivatives. Other suitable amphoteric and zwitterionic surfactants include those selected from the group consisting of betaines, sultaines, hydro-xysultaines, and branched and unbranched alkanoyl sarcosinates, and mixtures thereof.
[0137] In some embodiments of the present invention, personal care composition emulsions include a silicone containing emulsifier or surfactant. A wide variety of silicone emulsifiers find use herein. These silicone emulsifiers are typically organically modified organopolysiloxanes, also known to those skilled in the art as silicone surfactants. Useful silicone emulsifiers include dimethicone copolyols. These materials are polydimethyl siloxanes which have been modified to include polyether side chains such as polyethylene oxide chains, polypropylene oxide chains, mixtures of these chains and polyether chains containing moieties derived from both ethylene oxide and propylene oxide. Other examples include alkyl-modified dimethicone copolyols (i.e., compounds which contain C2-C30 pendant side chains). Still other useful dimethicone copolyols include materials having various cationic, anionic, amphoteric, and zwitterionic pendant moieties.
[0138] In further embodiments, the personal care compositions of the present invention contain at least one polymeric thickening agent. The polymeric thickening agents useful herein may have a number average molecular weight of greater than about 20,000, preferably greater than about 50,000, and more preferably greater than about 100,000. In some embodiments, the personal care compositions of the present invention comprise from about 0.01% to about 10%, preferably from about 0.1% to about 8%, and more preferably from about 0.25% to about 5% by weight of a polymeric thickening agent, or mixtures thereof.
[0139] Examples of polymer thickening agents for use herein include non-ionic thickening agents and anionic thickening agents or mixtures thereof. Suitable non-ionic thickening agents include polyacrylamide polymers, crosslinked poly(N-vinylpyrrolidones), polysaccharides, natural or synthetic gums, polyvinylpyrrolidone and polyvinylalcohol. Suitable anionic thickening agents include acrylic acid/ethyl acrylate copolymers, carboxyvinyl polymers and crosslinked copolymers of alkyl vinyl ethers and maleic anhydride. As an example, Noveon sells a thickener under the trademark of CARBOPOLยฎ resins or mixtures thereof. In some embodiments, suitable CARBOPOLยฎ resins are hydrophobically modified, while in other embodiments, suitable resins include those described in WO98/22085, or mixtures thereof.
[0140] In some embodiments, the personal care compositions of the present invention comprise at least one silicone oil phase. Silicone oil phase(s) generally comprise(s) from about 0.1% to about 20%, preferably from about 0.5% to about 10%, and more preferably from about 0.5% to about 5% by weight of the composition. In some embodiments, the silicone oil phase comprises one silicone component, while in alternative embodiments, the silicone oil phase comprises more than one silicone component.
[0141] In some embodiments, silicone components are fluids, including straight chain, branched and cyclic silicones. Suitable silicone fluids useful herein include silicones inclusive of polyalkyl siloxane fluids, polyaryl siloxane fluids, cyclic and linear polyalkylsiloxanes, polyalkoxylated silicones, amino and quaternary ammonium modified silicones, polyalkylaryl siloxanes or a polyether siloxane copolymer and mixtures thereof. In some embodiments, the silicone fluids are volatile, while in other embodiments, the silicone fluids are non-volatile. Silicone fluids generally have an average molecular weight of less than about 200,000. Suitable silicone fluids have a molecular weight of about 100,000 or less, preferably about 50,000 or less, and more preferably about 10,000 or less. In particularly preferred embodiments, the silicone fluid used in the present invention is selected from silicone fluids having a weight average molecular weight in the range from about 100 to about 50,000 and most preferably from about 200 to about 40,000.
[0142] Typically, silicone fluids that find use in the present invention have a viscosity ranging from about 0.65 to about 600,000 mm2s-1, and preferably from about 0.65 to about 10,000 mm2s-1 at 25ยฐ C. The viscosity can be measured by means of a glass capillary viscometer as set forth in Dow Corning Corporate Test Method CTM0004, Jul. 29, 1970. Suitable polydimethyl siloxanes that find use in the present invention include commercially available compounds (e.g., from General Electric Company and Dow Corning). In additional embodiments, essentially non-volatile polyalkylarylsiloxanes (e.g., polymethylphenylsiloxanes, having viscosities of about 0.65 to 30,000 mm2s-1 at 25ยฐ C.; available from General Electric Company and Dow Corning) find use in the present invention. Cyclic polydimethylsiloxanes suitable for use herein preferably have a ring structure incorporating from about 3 to about 7 (CH3)2SiO moieties, and preferably about 5 or more.
[0143] In some embodiments, silicone gums find use in the personal care compositions of the present invention. In some specific embodiments, a silicone oil phase comprises a silicone gum or a mixture of silicones including the silicone gum.
[0144] Typically, silicone gums have a viscosity at 25ยฐ C. in excess of about 1,000,000 mm2s-1. Silicone gums that find use in the present invention include dimethicones (e.g., those described in U.S. Pat. No. 4,152,416, herein incorporated by reference; Noll, Chemistry and Technology of Silicones, Academic Press, New York
[1968]; and General Electric Silicone Rubber Product Data Sheets SE 30, SE 33, SE 54 and SE 76). Specific examples of silicone gums include polydimethylsiloxane, (polydimethylsiloxane)(methylvinylsiloxane) copolymer, poly(dimethylsiloxane)-(diphenyl)(methylvinylsiloxane) copolymer and mixtures thereof. Preferred silicone gums for use herein include silicone gums having a molecular weight of from about 200,000 to about 4,000,000 selected from dimethiconol, dimethicone copolyol, dimethicone and mixtures thereof.
[0145] In some embodiments, the silicone phase herein comprises a silicone gum incorporated into the composition as part of a silicone gum-fluid blend. In some embodiments in which the silicone gum is incorporated as part of a silicone gum-fluid blend, the silicone gum constitutes from about 5% to about 40%, and preferably from about 10% to 20% by weight of the silicone gum-fluid blend. Suitable silicone gum-fluid blends herein are mixtures consisting essentially of:
[0146] (i) a silicone having a molecular weight of from about 200,000 to about 4,000,000 selected from dimethiconol, fluorosilicone and dimethicone and mixtures thereof; and
[0147] (ii) a carrier which is a silicone fluid, the carrier having a viscosity from about 0.65 mm2s-1 to about 100 mm2s-1, wherein the ratio of i) to ii) is from about 10:90 to about 20:80, and wherein said silicone gum-based component has a final viscosity of from about 100 mm2s-1 to about 100,000 mm2s-1, and more preferably from 500 mm2s-1 to about 10,000 mm2s-1.
[0148] Additional silicone components suitable for use in the silicone oil phase of some embodiments of the present invention include crosslinked polyorganosiloxane polymers, including those that are dispersed in a fluid carrier. In general, the crosslinked polyorganosiloxane polymers, together with their carrier (if present) comprise from about 0.1% to about 20%, preferably from about 0.5% to about 10%, and more preferably from about 0.5% to about 5% by weight of the personal care composition. Suitable polymers include those comprising polyorganosiloxane polymers crosslinked by a crosslinking agent (See, WO98/22085). Examples of suitable polyorganosiloxane polymers for use herein include methyl vinyl dimethicone, methyl vinyl diphenyl dimethicone and methyl vinyl phenyl methyl diphenyl dimethicone.
[0149] Another class of silicone components suitable for use in a silicone oil phase herein includes polydiorganosiloxane-polyoxyalkylene copolymers containing at least one polydiorganosiloxane segment and at least one polyoxyalkylene segment (See e.g., WO98/22085). Suitable polydiorganosiloxane-polyalkylene copolymers are available commercially under the tradename BELSILยฎ from Wacker-Chemie GmbH. An example of a copolymer fluid blend for use herein includes Dow Corning DC3225C which has the CTFA designation Dimethicone/Dimethicone copolyol.
[0150] In some embodiments, personal care compositions of the present invention also comprise at least one organic sunscreen. In some embodiments, suitable radioprotectives (e.g., sunscreens) exhibit UVA-absorbing properties and/or UVB-absorbing properties. The exact amount of the sunscreen active will vary, depending upon the desired Sun Protection Factor ("SPF") of the composition, as well as the desired level of UV protection. SPF is a commonly used indicator of photoprotection of a sunscreen against erythema. The SPF is defined as a ratio of the ultraviolet energy required to produce minimal erythema on protected skin to that required to produce the same minimal erythema on unprotected skin in the same individual. Amounts of the sunscreen may comprise from about 2% to about 20%, and preferably from about 4% to about 14% by weight of the personal care composition. Suitable sunscreens include, but are not limited to, those approved for use in the United States, Japan, Europe and Australia. In preferred embodiments, the compositions of the present invention comprise an SPF of about 2 to about 30, preferably about 4 about 30, and more preferably about 4 to about 15.
[0151] In some embodiments, the personal care compositions of the present invention include one or more UVA absorbing sunscreen actives that absorb UV radiation having a wavelength of from about 320 nm to about 400 nm. Suitable UVA absorbing sunscreen actives include those selected from dibenzoylmethane derivatives (See e.g., Lowe and Shaath (eds.), Sunscreens: Development, Evaluation, and Regulatory Aspects, Marcel Dekker, Inc
[1990]), anthranilate derivatives (e.g., methylanthranilate and homomethyl, 1-N-acetylanthranilate), and mixtures thereof. In some embodiments, the UVA absorbing sunscreen active is present in an amount suitable to provide broad spectrum UVA protection either independently, or in combination with, any other UV protective actives present in the composition.
[0152] Suitable UVA sunscreen actives include dibenzoylmethane sunscreen actives and their derivatives. They include, but are not limited to, those selected from 2-methyldibenzoylmethane, 4-methyldibenzoylmethane, 4-isopropyldibenzoylmethane, 4-tert-butyldibenzoylmethane, 2,4-dimethyldibenzoylmethane, 2,5-dimethyldibenzoylmethane, 4,4'-diisopropylbenzoylmethane, 4-(1,1-dimethylethyl)-4'-methoxydibenzoylmethane, 2-methyl-5-isopropyl-4'-methoxydibenzoylmethane, 2-methyl-5-tert-butyl-4'-methoxy-dibenzoylmethane, 2,4-dimethyl-4'-methoxydibenzoylmethane, 2,6-dimethyl-4'-tert-butyl-4'methoxydibenzoylmethane, and mixtures thereof. In one embodiment, the dibenzoyl sunscreen actives include those selected from 4-(1,1-dimethylethyl)-4'-methoxydibenzoylmethane, 4-isopropyldibenzoylmethane, and mixtures thereof. In further embodiments, the sunscreen active is 4-(1,1-dimethylethyl)-4'-methoxydibenzoylmethane.
[0153] The sunscreen active 4-(1,1-dimethylethyl)-4'-methoxydibenzoylmethane, which is also known as butyl methoxydibenzoylmethane or avobenzone, is commercially available under the names of PARSOLยฎ 1789 (Givaudan Roure (International) S. A.), and EUSOLEXยฎ 9020 (Merck & Co., Inc). The sunscreen 4-isoproplydibenzoylmethane, which is also known as isopropyldibenzoylmethane, is commercially available from Merck under the name of EUSOLEXยฎ 8020.
[0154] In some embodiments, the personal care compositions of the present invention further include one or more UVB sunscreen actives that absorb UV radiation having a wavelength of from about 290 nm to about 320 nm. The compositions comprise an amount of the UVB sunscreen active that is safe and effective in providing UVB protection either independently, or in combination with, any other UV protective actives present in the compositions. In some embodiments, the compositions comprise from about 0.1% to about 20%, preferably from about 0.1% to about 12%, and more preferably from about 0.5% to about 8% by weight of each UVB absorbing organic sunscreen, or as mandated by any relevant regulatory authority.
[0155] A variety of UVB sunscreen actives find use in the present invention, including, but not limited to organic sunscreen actives described in U.S. Pat. Nos. 5,087,372, 5,073,371, and 5,073,372, all of which are incorporated herein by reference. Additional sunscreens that find use in the present invention include those described in U.S. Pat. Nos. 4,937,370 and 4,999,186, both of which are incorporated herein by reference. Preferred UVB sunscreen actives are selected from 2-ethylhexyl-2-cyano-3,2-ethylhexyl N,N-dimethyl-p-aminobenzoate, p-amino-benzoic acid, oxybenzone, homomethyl salicylate, octyl salicylate, 4,4'-methoxy-t-butyldibenzoylmethane, 4-isopropyl dibenzoylmethane, 3-benzylidene camphor, 3-(4-methylbenzylidene)camphor, 3-diphenylacrylate, 2-phenyl-benzimidazole-5-sulphonic acid (PBSA), cinnamate esters and their derivatives such as 2-ethylhexyl-p-methoxycinnamate and octyl-p-methoxycinnamate, salicylate esters and their derivatives such as TEA triethanolamine salicylate, ethylhexyl saliycyilate, octyldimethyl para-aminobenzoic acidPABA, camphor derivatives and their derivatives, and mixtures thereof. Examples of organic sunscreen actives include 2-ethylhexyl-2-cyano-3,3-diphenylacrylate, 2-phenyl-benzimidazole-5-sulphonic acid (PBSA), octyl-p-methoxycinnamate, and mixtures thereof. Salt and acid neutralized forms of the acidic sunscreens are also find use herein.
[0156] In additional embodiments, at least one agent is added to the personal care compositions of the present invention to stabilize the UVA sunscreen(s) to prevent photo-degradation upon exposure to UV radiation, thereby maintaining UVA protection efficacy. A wide range of compounds are known to provide these stabilizing properties and should be chosen to complement both the UVA sunscreen and the composition as a whole. Suitable stabilizing agents include, but are not limited to, those described in WO 00/06110, and U.S. Pat. Nos. 5,972,316, 5,968,485, 5,935,556, 5,827,508, all of which are incorporated by reference. Examples of stabilizing agents for use in the present invention include 2-ethylhexyl-2-cyano-3,3-diphenylacrylate, ethyl-2-cyano-3,3-diphenylacrylate, 2-ethylhexyl-3,3-diphenylacrylate, ethyl-3,3-bis(4-methoxyphenyl)acrylate, diethylhexyl 2,6 napthalate and mixtures thereof (Symrise Chemical Company).
[0157] In further embodiments, at least one agent is added to any of the personal care compositions of the present invention to improve the skin substantivity of those compositions, particularly to enhance their resistance to being washed off by water or rubbed off. Examples include, but are not limited to, acrylates/C12-22 alkylmethacrylate copolymer, acrylate/acrylate copolymer, dimethicone, dimethiconol, graft-copoly (dimethylsiloxane/iI-butyl methacrylate), lauryl dimethicone, PVP/Hexadecane copolymer, PVP/Eicosene copolymer, tricontanyl PVP and trimethoxysiloxysiliacate.
[0158] In addition to organic sunscreens, some embodiments of personal care compositions of the present invention additionally comprise inorganic physical sunblocks, as known in the art (See e.g., CTFA International Cosmetic Ingredient Dictionary, 6th Edition,
[1995], pp. 1026-28 and 1103; Sayre et al., J. Soc. Cosmet. Chem., 41:103-109
[1990]; and Lowe et al., supra). In some particularly preferred embodiments, inorganic physical sunblocks such as zinc oxide and/or titanium dioxide, and mixtures thereof find use in the present invention.
[0159] In some particularly preferred embodiments of the present invention, physical sunblocks are present in an amount such that the present compositions are transparent on the skin (i.e., non-whitening), from about 0.5% to about 20%, preferably from about 0.51% to about 10%, and more preferably from about 0.5% to 5% by weight of the composition. Anatase, rutile, and/or amorphous titanium dioxide find use in the present invention. Manufacturers of micronized grade titanium dioxide and zinc oxide for sunscreen use include, but are not limited to Tayca Corporation, Uniqema, Shinetsu Chemical Corporation, Kerr-McGee, Nanophase, Nanosource, Sachtleben, Elementis, and BASF Corporation, as well as their distribution agents and those companies that further process the material for sunscreen use. In some embodiments, physical sunblock particles (e.g., titanium dioxide and zinc oxide), are uncoated, while in alternative embodiments, the particles are coated with a variety of materials including but not limited to amino acids, aluminium compounds such as alumina, aluminium stearate, aluminium laurate, and the like; carboxylic acids and their salts (e.g., stearic acid and its salts); phospholipids (e.g., lecithin); organic silicon compounds; inorganic silicon compounds (e.g., silica and silicates), and mixtures thereof. In some embodiments, the personal care compositions of the present invention contain from about 0.1% to about 15%, preferably from about 0.1% to about 7% and more preferably from about 0.5% to about 5% by weight of an inorganic sunscreen.
[0160] In some preferred embodiments, the personal care compositions of the present invention contain preservatives. Such preservatives include, but are not limited to pentylene glycol, ethylene diamine tetra acetate ("EDTA") and its salts, chlorhexidine (and its diacetate, dihydrochloride, digluconate derivatives), 1,1,1-trichloro-2-methyl-2-propanol, parachloro metaxylenol, polyhexamethylenebiguanide hydrochloride, dehydroacetic acid, diazolidinyl urea, 2,4-dichlorobenzyl alcohol, 4,4-dimethyl-1,3-oxazolidine, formaldehyde, glutaraldehyde, dimethylidantoin, imidazolidinyl urea, 5-chloro-2-methyl-4-isothiazolin-3-one, ortho-phenylphenol, 4-hydroxybenzoic acid and its (methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-) esters (also known as parabens), salts, trichlosan, 2-phenoxyethanol, phenyl mercuric acetate, borate, nitrate, quaternium-15, salicilate, salicylic acid and its salts, calcium, sorbic acid and its salts, iodopropanyl butylcarbamate, calcium sorbate, zinc pyrithione, benzyl alcohol, 5-bromo-5-nitro-1,3-dioxane, 2-bromo-2-nitropropane-1,3-diol, benzoic acid and its salts, sulfites, bisulfites, and benzalkonium chloride, phenoxyethanol and chloroxylenol, diazolidinyl urea, methylparaben, propylparaben, PG, isopropylparabens, isobutylparabens, butylparabens, ethylparaben, phenoxyethanol.
[0161] In further embodiments, a variety of optional ingredients such as neutralizing agents, perfumes and perfume solubilizing agents, and coloring agents, are included in the personal care compositions herein. Any additional ingredients should enhance the product, for example, the skin softness/smoothness benefits of the product. In addition, any such ingredients should not negatively impact the aesthetic properties of the product. Neutralizing agents suitable for use in neutralizing acidic group containing hydrophilic gelling agents herein include sodium hydroxide, potassium hydroxide, ammonium hydroxide, monoethanolamine, diethanolamine, amino methyl propanol, tris-buffer and triethanolamine.
[0162] In yet further embodiments, other optional materials are included in the present invention, including any of the various functional and/or active ingredients known to those skilled in the art. (See e.g., McCutcheon's Functional Materials, North American and International Editions, [2003)], published by MC Publishing Co.). Non-limiting examples include: keratolytic agents; water-soluble or solubilizable preservatives specifically at a level of from about 0.1% to about 5%, such as Germall 115, methyl, ethyl, propyl and butyl esters of hydroxybenzoic acid, benzyl alcohol, DMDM hydantoin iodopropanyl butylcarbanate available under the trade name GLYDANT PLUSยฎ (Lonza), EDTA, EUXYLยฎ K400, BROMOPOLยฎ (2-bromo-2-nitropropane-1,3-diol) and phenoxypropanol; anti-bacterials such as IRGASANยฎ and phenoxyethanol (specifically at levels of from about 0.1% to about 5%); soluble or colloidally-soluble moisturizing agents such as hyaluaronic acid and chondroitin sulfatestarch-grafted sodium polyacrylates such as SANWETยฎ IM-1000, IM-1500 and IM-2500 available from Celanese Superabsorbent Materials, Portsmith, Va., USA and described in U.S. Pat. No. 4,076,663; vitamins such as vitamin A, vitamin C, vitamin E, vitamin K and derivatives thereof and building blocks thereof; such as phytantriol; and vitamin K and components thereof such as the fatty alcohols such as dodecatrienol; alpha and beta hydroxyacids; aloe vera; sphingosines and phytosphingosines, cholesterol; skin whitening agents; N-acetyl cysteine; colouring agents; antibacterial agents such as TCC/TCS, also known as triclosan and trichlorocarbon; perfumes and perfume solubilizers. Examples of alpha hydroxy acids include glycolic acid, lactic acid, malic acid, and citric acid (whether derived synthetically or from natural sources and whether used alone or in combination), and their esters or relevant buffered combinations, such as glycolic acid in conjunction with ammonium glycolate. Other examples of alpha-hydroxy acids include: alpha-hydroxy ethanoic acid, alpha-hydroxyoctanoic acid, alpha-hydroxycaprylic acid, and hydroxycaprylic acid, mixed fruit acid, tri-alpha hydroxy fruit acids, triple fruit acid, sugar cane extract, alpha hydroxy and botanical comprise, 1-alpha hydroxy acid and glycomer in crosslinked fatty acids alpha nutrium. Specific examples of alpha hydroxy acids include glycolic acid and lactic acid. In a particular embodiment, alpha hydroxy acids are used in levels of up to about 10%.
[0163] In additional embodiments, optional materials such as pigments are included in the personal care compositions of the present invention, including water-insoluble pigments that contribute to and are included in the total level of oil phase ingredients. In some embodiments, the pigments used in the compositions of the present invention are organic, while in other embodiments they are inorganic. Also included within the term "pigment" are materials having a low color or luster, such as matte finishing agents, light scattering agents, and formulation aids (e.g., micas, seracites, and carbonate salts). Further examples of suitable pigments include titanium dioxide, predispersed titanium dioxide, iron oxides, zinc oxide, bismuth oxychloride (whether pre-dispersed and/or pre-coated or not) coated iron oxides, ultramarine blue, D&C dyes and lakes, FD&C colors, natural color additives such as carmine, and mixtures thereof. Depending upon the type of composition, a mixture of pigments is usually used. Exemplary pigments for use herein from the viewpoint of moisturization, skin feel, skin appearance and emulsion compatibility are treated pigments (e.g., pigments treated with compounds, including but not limited to amino acids, silicones, lecithin and ester oils).
[0164] In most embodiments, the pH of the personal care compositions herein is in the range from about 3.5 to about 10, preferably from about 4 to about 8, and more preferably from about 5 to about 7, wherein the pH of the final composition is adjusted by addition of acidic, basic or buffer salts as necessary, depending upon the composition of the forms and the pH-requirements of the compounds.
[0165] Those skilled in the art will appreciate the various techniques for preparing the personal care compositions of the present invention, any of which may be employed herein. In general the aqueous phase and/or the oil phases are prepared separately, with materials of similar phase partitioning being added in any suitable order. For emulsion final products, the two phases are combined with vigorous stirring and/or homogenization as necessary to reduce the size of the internal phase droplets. Any ingredients in the formulation with high volatility, or which are susceptible to hydrolysis or decomposition at high temperatures, are preferably added with gentle stirring towards the end of the process, and/or at the post emulsification stage, if applicable. As known to those skilled in the art, dosage frequency and amount depends upon the desired performance criteria.
EXPERIMENTAL
[0166] The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
[0167] In the experimental disclosure which follows, the following abbreviations apply: M (molar); mM (millimolar); ฮผM (micromolar); nM (nanomolar); mol (moles); mmol (millimoles); ฮผmol (micromoles); nmol (nanomoles); gm (grams); mg (milligrams); ฮผg (micrograms); pg (picograms); L (liters); ml (milliliters); ฮผl (microliters); cm (centimeters); mm (millimeters); ฮผm (micrometers); nm (nanometers); ยฐ C. (degrees Centigrade); Tg (glass transition temperature); DI (distilled water); MQ (milli-Q water; deionized water with further volatiles removed, to provide pure water with an electrical resistance of 18.2 ohms); PSI (pounds per square inch); CER (carbon dioxide evolution rate); cDNA (copy or complimentary DNA); DNA (deoxyribonucleic acid); ssDNA (single stranded DNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotide triphosphate); RNA (ribonucleic acid); PBS (phosphate buffered saline); g (gravity); xg (times gravity); OD (optical density); Dulbecco's phosphate buffered solution (DPBS); HEPES (N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); HBS (HEPES buffered saline); SDS (sodium dodecylsulfate); Tris-HCl (tris[Hydroxymethyl]aminomethane-hydrochloride); DMSO (dimethyl sulfoxide); PEI (polyethyleneimine); EGTA (ethylene glycol-bis(B-aminoethyl ether) N,N,N',N'-tetraacetic acid); EDTA (ethylenediaminetetracetic acid); FBS (fetal bovine serum); FGM (fibroblast growth media); DMEM (Dulbecco's Modified Essential Media); DPBS (Dulbecco's phosphate buffered solution; PPTI (Protein Polymer Technologies, Inc., San Diego, Calif.); Dow Corning (Dow Corning Corporation, Midland, Mich.); Clariant (Clariant Corporation, Charlotte, N.C.); R&D (R&D Systems, Inc., Minneapolis, Minn.); Takara (Takara USA Corporation., New York, N.Y.); Biocolor (Biocolor, Ltd., Newtownabbey, Ireland); Hercules (Hercules, Inc., Wilmington, Del.); Cognis (Cognis Corp., Hoboken, N.J.); Uniqema (Uniqema, Inc., Wilmington, Del.); Croda (Croda, Inc., Parsippany, N.J.); Lonza (Lonza, Inc., Fairlawn, N.J.); Sun (Sun Chemical, Inc., Fort Lee, N.J.); Aqualon (Aqualon, Wilmington, Del.); S&P (Strahl & Ptisch, W. Babylon, N.Y.); Color Techniques (Color Techniques, South Plainfield, N.J.); Cascade Biologics (Cascade Biologics, Portland, Oreg.); ATCC (American Type Culture Collection, Rockville, Md.); Gibco/BRL (Gibco/BRL, Grand Island, N.Y.); Sigma (Sigma Chemical Co., St. Louis, Mo.); Pharmacia (Pharmacia Biotech, Piscataway, N.J.); Invitrogen (Invitrogen, Inc., Grand Island, N.Y.); Abbott (Abbott Laboratories, Abbott Park, Ill.); Perkin Elmer (PerkinElmer Life Sciences, Boston Mass.); and Stratagene (Stratagene, La Jolla, Calif.).
[0168] In the experiments described below, the "RSPP test materials" are indicated in the following Table:
TABLE-US-00001 TABLE 1 Test Materials RSPP Designation RSPP GC2596-26-A SELP47K (monodispersed) GC2596-26-B SELP47K (polydispersed) GC2596-26-C SELP47K (monomeric form generated by lysC treatment) GC2596-26-D SELP47K (SELP47K-P4 RSPP) GC2596-26-E DCP6 RSPP (collagen-like protein)
[0169] Material GC2596-26-A was monodispersed 13-mer SELP47K RSPP (See, Example 1), while Material GC2596-26-B was polydispersed SELP47K (See, Example 2). Material GC2596-26-C was SELP47K that was enzymatically treated with lysC to cleave full-length SELP47K into its monomeric unit having lysine residues at each end (See, Example 3). Material GC2596-26-D was SELP47K modified with two units of ALSYP (SEQ ID NO:36) peptides placed in each SELP47K unit prior to silk peptide repeats. (See, Example 4) Material GC2596-26-E was a modified collagen repeat sequence that was obtained from Protein Polymer Technologies (See, Example 5).
Example 1
Isolation of SELP
[0170] In this Example, experiments conducted to isolate and purify a genetically engineered silk-elastin repeat sequence protein block copolymer (SELP) from E. coli are described. E. coli containing a specific silk-elastin repeat sequence protein copolymer SELP47K (GC2596-26-A) recombinant DNA was obtained from PPTI. The E. coli may be prepared in accordance with the methods described in U.S. Pat. Nos. 5,243,038 and 6,355,776, incorporated herein by reference. The recovery of kilogram quantities of SELP was also demonstrated. The silk-elastin copolymer SELP47K had a general structure of head-[(GAGAGS)2(GVGVP)3GKGVP(GVGP)4(GAGAGS)2]13-- tail (SEQ ID NO:19). The copolymer contained 886 amino acids, with 780 amino acids in the repeating sequence unit. The SELP47K had a molecular weight of about 70,000 Daltons, and the pI of the protein is 10.5.
[0171] Monodispersed silk-elastin protein polymer SELP47K was produced for application testing in the following manner. E. coli fermentation was performed to produce a cell-paste containing monodispersed SELP47K. The cell-paste was placed in ice cold water and homogenized to make the cell extract. The cell-extract was mixed with polyethyleneimine and a filter-aid and was allowed to stir at 7ยฐ C. for one hour. The polyethyeleneimine caused precipitation of cell debris and a significant amount of E. coli proteins. The SELP47K containing reaction mixture was then filtered using Rotary Drum Vacuum Filter (RVDF). The filtered SELP47K solution was then mixed with ammonium sulfate to 25% saturation, which led to precipitation of SELP47K. Precipitated SELP47K and mother liquor was mixed with a filter-aid and again filtered using RVDF. The RVDF cake containing SELP47K and filter-aid was mixed with cold water to dissolve the SELP47K. This precipitation and solubilization step was repeated one more time to improve the purity profile of the SELP47K. Purified monodispersed SELP47K was then water-exchanged until the conductivity of SELP solution reached 50 ฮผS/cm2. The monodispersed SELP solution was then concentrated to 10% wt/vol and then lyophilized to make powdered monodispersed SELP47K protein polymer. The material was stored at -70ยฐ C. until needed for application testing.
[0172] SELP variants were either obtained from PPTI or genetically engineered. Table 2 provides a list of the variants and their properties.
TABLE-US-00002 TABLE 2 SELP Variants and Their Properties. Number Molecular Variant of Lysine Weight Isoelectric Name Subunits Substitution (Da) Point SELP47E 13 Glutamic Acid 70,212 4.16 SELP47K-3 3 none 20,748 9.52 SELP47R-3 3 Arginine 20,960 10.5 SELP47E-3 3 Glutamic Acid 20,879 5.9 SELP27K 13 none 59,401 10.53 SELP37K 13 none 64,605 10.53 SELP58 13 none 74,765 6.7 SELP67K 13 none 80,347 10.53
[0173] The E. coli strains containing a specific silk-elastin repeat sequence protein copolymer SELP47K, SELP37K, and SELP27K recombinant DNA were also obtained from PPTI. SELP67K, SELP58, SELP37K and SELP27K variant proteins were produced in 14 L fed batch culture using standard SELP47K production protocols, as described above. Proteins were purified and characterized as follows: 40 grams of cell pastes collected from 14L cultures were lysed via French-press followed by the addition of polyethyleneimine (0.8 w/v %). Centrifugation was used to separate the cellular debris from the cell extract. SELP polymers were precipitated from the cell extract using ammonium sulfate (30% saturation), collected by centrifugation and reconstituted in water. Residual salts were removed by dialysis against water and SELP polymers were lyophilized and characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SELP47K-3 species was excised from SDS-PAGE gels and further characterized, its identity confirmed, by LC-MS/MS (Liquid Chromatographic Mass Spectroscopy). The molecular weight of the intact SELP47K-3 protein was also confirmed using MALDI-TOF/MS (Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry).
[0174] The protocol used for the genetic engineering of variants SELP47E, SELP47K-3, SELP47R-3, and SELP47E-3 is a modification of a commercially available kit designed to create single base pair changes in multiple sites along a particular DNA sequence (QUIKCHANGEยฎ Multi-Site-Directed Mutagenesis Kit, Stratagene #200513). The standard protocol involves the construction of single direction 5' phosphorylated primers that will hybridize to plasmid template regions of interest and incorporate point mutations. Thermocycling is employed that includes a ligation reaction designed to link the multiple primers during each round of synthesis.
[0175] SELP DNA sequences are unique in that the multiple repeating subunits are identical. In order to change a single amino acid residue in all subunits a single change is effectively performed multiple times. The above protocol was further modified in that primers were designed pair-wise, complementary, thereby creating PCR amplification conditions in the thermocycling process. Amplified plasmid DNA was then used to transform E. coli cells and can be further screened and characterized for desired mutations.
Conversion of SELP Lysine Residues.
[0176] Primers were designed that direct a single base change mutation resulting in conversion of lysine residues to glutamic acids or arginines while simultaneously creating a unique restriction enzyme site at this location used for subsequent plasmid screening. 5' phosphorylated primers were made complementary, in both directions (both strands) as follows:
[0177] Glutamic Acid conversion:
TABLE-US-00003 (SEQ ID NO: 21) 5'-GGGAGTTGGTGTACCTGGAGAAGGTGTTCCGGGGGTAGG-3' (SEQ ID NO: 22) 3'-CCCTCAACCACATGGACCTCTTCCACAAGGCCCCCATCC-5'
[0178] (A20 was converted to G20)
[0179] Arginine Conversion:
TABLE-US-00004 (SEQ ID NO: 23) 5'-GGGAGTTGGGGTACCTGGACGAGGTGTTCCGGGGGTAGG-3' (SEQ ID NO: 24) 3'-CCCTCAACCCCATGGACCTCGAGGTGGAACCCCCCCATCC-5'
[0180] (G19 and T20 were converted to C and G)
[0181] The QUIKCHANGEยฎ Multi-Site kit was used per the manufacturer's protocol except that both complementary primers were included. Five ฮผl of each reaction was used to transform TOP10 cells as per protocol (Invitrogen). 100 ฮผl of salt optimized carbon (SOC) outgrowth were plated per reaction. Transformants were picked and grown in 5 ml LB containing 50 ppm kanamycin. Plasmid DNA was obtained from cultures using the Qiagen plasmid miniprep kit and analyzed by digestion with appropriate restriction enzymes followed by gel electrophoresis. Constructs that appeared correct were confirmed by DNA sequencing. Several rounds of the above protocol were required to obtain the SELP47E variant. In all cases this method resulted in the creation of a library consisting of variants spanning a range of subunits. This distribution ranged from 1 to 17 subunits. SELP47E-3 and SELP47R-3 were a result of this distribution. SELP47K-3 resulted from using the above methods to convert SELP47E-3 glutamic acids back to lysines.
[0182] Successful construct plasmids were used to transform E. coli MM294 using Lauryl Bertani (LB) plates containing 50 ppm kanamycin. Single colonies were picked and grown in 60 ml TM2 (recipe)+2% glucose, 50 ppm kanamycin in 500 ml fluted Erlenmeyer flasks, 30ยฐ C., 250 rpm, 16 hrs. Cell culture was supplemented with glycerol (10% v/v), and 1.5 ml aliquots were placed in cryovials and stored at -80ยฐ C. Random vials were tested for contamination by incubating 10 ฮผl inoculating loopfuls on LA+1.6% skim milk plates, 37ยฐ C., for 16 hrs. Integrity of the plasmids was also confirmed using plasmid purification and analysis using restriction enzyme digestion/gel electrophoresis as well as DNA sequencing. Frozen cryovials were prepared using methods known in the art and used as seed stocks for subsequent culturing, protein production.
[0183] SELP47K-3, SELP47E-3 and SELP47R-3 variant proteins were produced in 14 L fed batch culture using standard SELP47K production protocols used above. Proteins were purified and characterized as follows: 40 grams of cell pastes collected from 14L cultures were lysed via French-press followed by the addition of polyethyleneimine (0.8 w/v %). Centrifugation was used to separate the cellular debris from the cell extract. SELP polymers were precipitated from the cell extract using ammonium sulfate (30% saturation), collected by centrifugation and reconstituted in water. Residual salts were removed by dialysis against water and SELP polymers were lyophilized and characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The SELP47K-3 species was excised from SDS-PAGE gels and further characterized, its identity confirmed, by LC-MS/MS. The molecular weight of the intact SELP47K-3 protein was also confirmed using MALDI-TOF/MS.-M3H+1641, M4H+1231 of silk-elastin unit at ห5 kDa.
Example 2
Preparation of Polydispersed SELP47K for Application Testing
[0184] Experiments involving the purification and preparation of the polydispersed SELP47K silk-elastin protein polymer (GC2596-26-B) for application testing are described in this Example. Cell separation from the fermentation broth was done using microfiltration. A cell disruption to make a cell-extract was done using a French-press. The cell extract was separated from the cell-debris using polyethyleneimine and a filter-aid. The cell extract was mixed with ammonium sulfate to 25% saturation to precipitate the silk-elastin protein polymer. The precipitated silk-elastin protein polymer was further purified by dissolving it in water and reprecipitating it with ammonium sulfate.
[0185] In order to prepare a polydispersed silk-elastin protein polymer, the precipitated silk-elastin protein polymer was again dissolved in water and mixed with a trace amount of protease (BPN'Y217L) (Genencor International). The protease was then inactivated and destroyed by acid treatment. The polydispersed silk-elastin protein polymer was then ultrafiltered until the silk-elastin protein polymer solution reached an electrical conductivity of 50 ฮผS/m2.
[0186] The polydispersed silk-elastin protein polymer solution was concentrated to 10 wt % and was lyophilized. The lyophilized polydisperesed silk-elastin protein polymer (GC2596-26-B) powder was stored at -70ยฐ C. until use. The lyophilized polydispersed silk-elastion protein was then dissolved in deionized water to a desired concentration for skin and hair application testing.
Example 3
Cleavage of Monodispersed SELP47K
[0187] This Example describes the methods used to cleave monodispersed SELP47K to its monomeric unit. The purification and formation of monomeric unit of SELP47K (4920 kDA molecular weight) (GC2596-26-C) was carried out using monodispersed material of SELP47K produced as in Example 1. The monodispersed SELP47K was dissolved in water and was treated with endopeptidase lysC protease (Sigma) specific for cleaving protein at lysine residue for 30 minutes at room temperature. The lysC protease was then inactivated and destroyed by acid treatment. The monomeric unit of SELP47K was then ultra-filtered until protein polymer solution conductivity reached 50 ฮผS/m2. The monomeric unit of SELP47K solution was then lyophilized to get the solid material and stored at -70 C prior to application testing.
Example 4
SELP47K-P4 Biosynthesis and Production
[0188] In this Example, the construction, analysis and verification of the SELP47K-P4 RSPP is described. The amino acid sequence for this RSPP is:
TABLE-US-00005 (SEQ ID NO: 32) MDPVVLQRRD WENPGVTQLN RLAAHPPFAS DPMGAGAGSG AGAGSALSYP GVGVPGVGVP GVGVPGVGVP GKGVPGVGVP GVGVPGVGVP ALSYPGAGAG SGAGAGSGAG AGSGAGAGSA LSYPGVGVPG VGVPGVGVPG VGVPGKGVPG VGVPGVGVPG VGVPALSYPG AGAGSGAGAG SGAGAGSGAG AGSALSYPGV GVPGVGVPGV GVPGVGVPGK GVPGVGVPGV GVPGVGVPAL SYPGAGAGSG AGAGSGAGAG SGAGAGSALS YPGVGVPGVG VPGVGVPGVG VPGKGVPGVG VPGVGVPGVG VPALSYPGAG AGSGAGAGSG AGAGSGAGAG SALSYPGVGV PGVGVPGVGV PGVGVPGKGV PGVGVPGVGV PGVGVPALSY PGAGAGSGAG AGSGAGAGSG AGAGSALSYP GVGVPGVGVP GVGVPGVGVP GKGVPGVGVP GVGVPGVGVP ALSYPGAGAG SGAGAGSGAG AGSGAGAGSA LSYPGVGVPG VGVPGVGVPG VGVPGKGVPG VGVPGVGVPG VGVPALSYPG AGAGSGAGAG SGAGAGSGAG AGSALSYPGV GVPGVGVPGV GVPGVGVPGK GVPGVGVPGV GVPGVGVPAL SYPGAGAGSG AGAGSGAGAG SGAGAGSALS YPGVGVPGVG VPGVGVPGVG VPGKGVPGVG VPGVGVPGVG VPALSYPGAG AGSGAGAGSG AGAGSGAGAG SALSYPGVGV PGVGVPGVGV PGVGVPGKGV PGVGVPGVGV PGVGVPALSY PGAGAGSGAG AGSGAGAGSG AGAGSALSYP GVGVPGVGVP GVGVPGVGVP GKGVPGVGVP GVGVPGVGVP ALSYPGAGAG SGAGAGSGAG AGSGAGAGSA LSYPGVGVPG VGVPGVGVPG VGVPGKGVPG VGVPGVGVPG VGVPALSYPG AGAGSGAGAG SGAGAGSGAG AGSALSYPGV GVPGVGVPGV GVPGVGVPGK GVPGVGVPGV GVPGVGVPAL SYPGAGAGSG AGAGSGAGAM DPGRYQDLRS HHHHHH*
[0189] The DNA sequence for this RSPP is:
TABLE-US-00006 (SEQ ID NO: 33) ATGGATCCCG TCGTTTTACA ACGTCGTGAC TGGGAAAACC CTGGCGTTAC CCAACTTAAT CGCCTTGCAG CACATCCCCC TTTCGCCAGC GATCCGATGG GAGCGGGTGC CGGTTCTGGT GCAGGCGCGG GCTCTGCGCT GAGCTATCCG GGTGTTGGAG TGCCAGGTGT CGGTGTTCCG GGTGTAGGCG TTCCGGGAGT TGGTGTACCT GGAAAAGGTG TTCCGGGGGT AGGTGTGCCG GGCGTTGGAG TACCAGGTGT AGGCGTCCCG GCGCTGAGCT ATCCGGGAGC GGGTGCTGGT AGCGGCGCAG GCGCGGGCTC TGGAGCGGGT GCCGGTTCTG GTGCAGGCGC GGGCTCTGCG CTGAGCTATC CGGGTGTTGG AGTGCCAGGT GTCGGTGTTC CGGGTGTAGG CGTTCCGGGA GTTGGTGTAC CTGGAAAAGG TGTTCCGGGG GTAGGTGTGC CGGGCGTTGG AGTACCAGGT GTAGGCGTCC CGGCGCTGAG CTATCCGGGA GCGGGTGCTG GTAGCGGCGC AGGCGCGGGC TCTGGAGCGG GTGCCGGTTC TGGTGCAGGC GCGGGCTCTG CGCTGAGCTA TCCGGGTGTT GGAGTGCCAG GTGTCGGTGT TCCGGGTGTA GGCGTTCCGG GAGTTGGTGT ACCTGGAAAA GGTGTTCCGG GGGTAGGTGT GCCGGGCGTT GGAGTACCAG GTGTAGGCGT CCCGGCGCTG AGCTATCCGG GAGCGGGTGC TGGTAGCGGC GCAGGCGCGG GCTCTGGAGC GGGTGCCGGT TCTGGTGCAG GCGCGGGCTC TGCGCTGAGC TATCCGGGTG TTGGAGTGCC AGGTGTCGGT GTTCCGGGTG TAGGCGTTCC GGGAGTTGGT GTACCTGGAA AAGGTGTTCC GGGGGTAGGT GTGCCGGGCG TTGGAGTACC AGGTGTAGGC GTCCCGGCGC TGAGCTATCC GGGAGCGGGT GCTGGTAGCG GCGCAGGCGC GGGCTCTGGA GCGGGTGCCG GTTCTGGTGC AGGCGCGGGC TCTGCGCTGA GCTATCCGGG TGTTGGAGTG CCAGGTGTCG GTGTTCCGGG TGTAGGCGTT CCGGGAGTTG GTGTACCTGG AAAAGGTGTT CCGGGGGTAG GTGTGCCGGG CGTTGGAGTA CCAGGTGTAG GCGTCCCGGC GCTGAGCTAT CCGGGAGCGG GTGCTGGTAG CGGCGCAGGC GCGGGCTCTG GAGCGGGTGC CGGTTCTGGT GCAGGCGCGG GCTCTGCGCT GAGCTATCCG GGTGTTGGAG TGCCAGGTGT CGGTGTTCCG GGTGTAGGCG TTCCGGGAGT TGGTGTACCT GGAAAAGGTG TTCCGGGGGT AGGTGTGCCG GGCGTTGGAG TACCAGGTGT AGGCGTCCCG GCGCTGAGCT ATCCGGGAGC GGGTGCTGGT AGCGGCGCAG GCGCGGGCTC TGGAGCGGGT GCCGGTTCTG GTGCAGGCGC GGGCTCTGCG CTGAGCTATC CGGGTGTTGG AGTGCCAGGT GTCGGTGTTC CGGGTGTAGG CGTTCCGGGA GTTGGTGTAC CTGGAAAAGG TGTTCCGGGG GTAGGTGTGC CGGGCGTTGG AGTACCAGGT GTAGGCGTCC CGGCGCTGAG CTATCCGGGA GCGGGTGCTG GTAGCGGCGC AGGCGCGGGC TCTGGAGCGG GTGCCGGTTC TGGTGCAGGC GCGGGCTCTG CGCTGAGCTA TCCGGGTGTT GGAGTGCCAG GTGTCGGTGT TCCGGGTGTA GGCGTTCCGG GAGTTGGTGT ACCTGGAAAA GGTGTTCCGG GGGTAGGTGT GCCGGGCGTT GGAGTACCAG GTGTAGGCGT CCCGGCGCTG AGCTATCCGG GAGCGGGTGC TGGTAGCGGC GCAGGCGCGG GCTCTGGAGC GGGTGCCGGT TCTGGTGCAG GCGCGGGCTC TGCGCTGAGC TATCCGGGTG TTGGAGTGCC AGGTGTCGGT GTTCCGGGTG TAGGCGTTCC GGGAGTTGGT GTACCTGGAA AAGGTGTTCC GGGGGTAGGT GTGCCGGGCG TTGGAGTACC AGGTGTAGGC GTCCCGGCGC TGAGCTATCC GGGAGCGGGT GCTGGTAGCG GCGCAGGCGC GGGCTCTGGA GCGGGTGCCG GTTCTGGTGC AGGCGCGGGC TCTGCGCTGA GCTATCCGGG TGTTGGAGTG CCAGGTGTCG GTGTTCCGGG TGTAGGCGTT CCGGGAGTTG GTGTACCTGG AAAAGGTGTT CCGGGGGTAG GTGTGCCGGG CGTTGGAGTA CCAGGTGTAG GCGTCCCGGC GCTGAGCTAT CCGGGAGCGG GTGCTGGTAG CGGCGCAGGC GCGGGCTCTG GAGCGGGTGC CGGTTCTGGT GCAGGCGCGG GCTCTGCGCT GAGCTATCCG GGTGTTGGAG TGCCAGGTGT CGGTGTTCCG GGTGTAGGCG TTCCGGGAGT TGGTGTACCT GGAAAAGGTG TTCCGGGGGT AGGTGTGCCG GGCGTTGGAG TACCAGGTGT AGGCGTCCCG GCGCTGAGCT ATCCGGGAGC GGGTGCTGGT AGCGGCGCAG GCGCGGGCTC TGGAGCGGGT GCCGGTTCTG GTGCAGGCGC GGGCTCTGCG CTGAGCTATC CGGGTGTTGG AGTGCCAGGT GTCGGTGTTC CGGGTGTAGG CGTTCCGGGA GTTGGTGTAC CTGGAAAAGG TGTTCCGGGG GTAGGTGTGC CGGGCGTTGG AGTACCAGGT GTAGGCGTCC CGGCGCTGAG CTATCCGGGA GCGGGTGCTG GTAGCGGCGC AGGCGCGGGC TCTGGAGCGG GTGCCGGTTC TGGTGCAGGC GCGGGCTCTG CGCTGAGCTA TCCGGGTGTT GGAGTGCCAG GTGTCGGTGT TCCGGGTGTA GGCGTTCCGG GAGTTGGTGT ACCTGGAAAA GGTGTTCCGG GGGTAGGTGT GCCGGGCGTT GGAGTACCAG GTGTAGGCGT CCCGGCGCTG AGCTATCCGG GAGCGGGTGC TGGTAGCGGC GCAGGCGCGG GCTCTGGAGC GGGTGCCATG GACCCGGGTC GATATCAGGA TCTTAGATCT CATCACCATC ACCATCACTA A
[0190] SELP constructs in which all polymer subunits contain additional P4 (ALSYP; SEQ ID NO:36) peptide sequences were made as follows: Artificial gene synthesis was used to create a 2 subunit version of the desired construct in which each identical subunit contained a unique non-palindromic restriction site. Specifically, SELP subunits contained either BBI (Bowman-Birk Inhibitor)-derived P4 sequence (i.e., ALSYP; SEQ ID NO:36) incorporated in two places per subunit, flanking the silk region, incorporated between the C-terminal end of the silk region and the N-terminal end of the elastin region. A BsgI restriction enzyme cleavage site was designed within each subunit.
[0191] The construct DNA was digested with BsgI, which resulted in the release of a single subunit per molecule. Resulting modified SELP monomers as well as plasmid vector were purified from agarose gels and concentrated using a Spin Vac. Monomers were self-ligated using T4 DNA ligase (New England Biolabs) overnight at 16ยฐ C., as per the manufacturer's instructions. Original purified vector was added to the ligation mix with additional fresh ligase and incubated at 16ยฐ C. for an additional 24 hours. This ligation mix was used to transform E. coli TOP10 cells (Invitrogen) as per the manufacturer's instructions. The resulting transformants were used to inoculate culture tubes containing 5 ml Luria broth and appropriate antibiotic. Tubes were incubated for 16 hrs, at 37ยฐ C., and with shaking at 250 rpm. Plasmids were purified from resulting cultures using a Plasmid Miniprep kit (Qiagen). Complete SELP inserts were excised from vector by digestion with EcoRV and BamHI restriction enzymes and sized using agarose gel elecrophoresis in order to identify constructs containing the desired 13 total subunits.
[0192] Appropriate SELP-P4 gene constructs were subcloned into a vector containing the lambda Pr promoter and transformed into production the host E. coli mM294. Prepared seed vials were stored at -80ยฐ C. These seed vials were used for fermentation of SELP47K-P4 repeat sequence protein polymers. The strain tested for fermentation run was MM294/pSELP01472 (SELP47K-P4; GC2596-26-D). The E. coli strain was incubated in defined TM2 medium and batched glucose. The fermentation was continued with a feed of 2 g/min of glucose upon consumption of batched glucose until the cells in the fermenters grew up to an OD of 60. The expression of SELP47K protein polymer was started with temperature induction by raising the fermentation temperature to 40ยฐ C. The fermentation and growth rates were very similar to that of SELP47K (Example 1). The cells grew well prior to, as well as after induction. Cell pastes were harvested by centrifuging fermentation broth after 20% drop in CER and with no further rise in OD. Purification of SELP47K-P4 RSPP test material was performed by first taking the cell paste in 1:2 ratio in DI water, followed by homogenizing the cells using French-Press at 8000 PSI. Homogenized cells were mixed with 0.1%-10% PEI (polyethyleneimine) to flock-out cell debris. The cell debris was removed by centrifugation. The cell extract generated from centrifugation was mixed with ammonium sulfate (10-25% saturation) to precipitate SELP47K-P4 RSPP test material. Precipitated SELP47K-P4 RSPP was separated from the solution and dissolved in either MQ water or a chaotropic agent (e.g., urea or guanidinium hydrochloride). SELP47K-P4 RSPP was purified by dialysis and then lyophilized as solid powder for storage, using methods known in the art. Analysis of purified P4-SELP47K was carried out using SDS gel electrophoresis as well as mass spectrometry to confirm the formation of fusion polymer.
Example 5
Production of DCP6 (GC 2596-26-E)
[0193] In this Example, experiments conducted to isolate and purify a genetically engineered collagen-like repeat sequence protein block copolymer (DCP6) from E. coli are described. E. coli host MM294/pPPTI0301 strain containing a specific silk-elastin repeat sequence protein copolymer DCP6 (GC2596-26-E) recombinant DNA was obtained from PPTI. The E. coli was prepared in accordance with the methods described in U.S. Pat. Nos. 5,243,038 and 6,355,776, incorporated by reference herein.
[0194] The collagen-like repeat sequence protein polymer had a general structure of head-[(GAHGPAGPK)2(GAQGPAGPG)24(GAHGPAGPK)2]4-tail (SEQ ID NO:37). The copolymer contained 1008 amino acids, with 947 amino acids in the repeating sequence units. The DCP6 had a molecular weight of about 78,881 Daltons (calculated using mass spectrometry), and the pI of the protein was 11.18. The amino acid sequence is shown below:
TABLE-US-00007 (SEQ ID NO: 34) MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPMGAHGPAGPKGAHGP AGPKGAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGP AGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGP AGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGP AGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGP AGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGP GGAHGP AGPKGAHGPAGPK GAHGPAGPKGAHGPAGPKGAQGPAGPG GAQGPA GPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPA GPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPA GPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPA GPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPA GPG GAQGPAGPG GAQGPAGPGGAHGPAGPKGAHGPAGPKGAHGPA GPKGAHGPAGPKGAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPA GPG GAHGPAGPKGAHGPAGPKGAHGPAGPKGAHGPAGPKGAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPG GAQGPAGPGGAHGPAGPKGAHGPAG PKMDPGRYQLSAGRYHYQLVWCQK
[0195] The DNA sequence of this RSPP is
TABLE-US-00008 (SEQ ID NO: 35) GGCGCGCATGGCCCGGCGGGCCCGAAAGGCGCGCATGGCCCGGCGGGCC CGAAAGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGC GGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGC CCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGC AGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGG CGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCG GGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGG GCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCC GGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAG GGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCG CGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGG CGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGC CCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGG CGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGG CCCGGCGGGCCCGGGCGGCGCGCATGGCCCGGCGGGCCCGAAAGGCGCG CATGGCC CGGCGGGCCCGAAAGGCGCGCATGGCCCGGCGGGCCCGAAAGGCGCGCA TGGCCCGGCGGGCCCGAAAGGCGCGCAGGGCCCGGCGGGCCCGGGCGGC GCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGG GCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGG CCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCG GCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGG GCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGC GCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGC GGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCC CGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGC GGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGC CCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGC AGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGG CGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCG GGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCATGGCCCGGCGG GCCCGAAAGGCGCGCATGGCCCGGCGGGCCCGAAAGGCGCGCATGGCCC GGCGGGCCCGAAAGGCGCGCATGGCCCGGCGGGCCCGAAAGGCGCGCAG GGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCG CGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGG CGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGC CCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGG CGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGG CCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCG CAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCG GCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCC GGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCG GGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCC CGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCA GGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGC GCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGG GCGGCGCGCATGGCCCGGCGGGCCCGAAAGGCGCGCATGGCCCGGCGGG CCCGAAAGGCGCGCATGGCCCGGCGGGCCCGAAAGGCGCGCATGGCCCG GCGGGCCCGAAAGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGG GCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGC GCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGC GGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCC CGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGC GGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGC CCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGC AGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGG CGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCG GGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGG GCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCC GGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCGCGCAG GGCCCGGCGGGCCCGGGCGGCGCGCAGGGCCCGGCGGGCCCGGGCGGCG CGCAGGGCCCGGCGGGCCCGGGCGGCGCGCATGGCCCGGCGGGCCCGAA AGGCGCGCATGGCCCGGCGGGCCCGAAA
[0196] DCP6 RSPP test material was produced for application testing in the following manner. The strain tested for fermentation run was MM294/pPPTI301 (DCP6; GC2596-26-E). The E. coli strain was incubated in defined TM2 medium and batched glucose. The fermentation was continued with a feed of 2 g/min of glucose upon consumption of batched glucose until the cells in the fermenters grew up to an OD (550 nm) of 60. The expression of SELP47K protein polymer was started with temperature induction by raising the fermentation temperature to 40ยฐ C. The fermentation and growth rates were very similar to that of SELP47K (Example 1). The cells grew well prior to, as well as after induction. Cell pastes were harvested by centrifuging fermentation broth after 20% drop in CER and with no further rise in OD (550 nm). The cell-paste was placed in DI water and homogenized to make the cell extract. The cell-extract was centrifuged and lysate was dialysed against pH 4 formate buffer followed by neutralization of dialysate. The dialysate was further purified using bulk anion exchange chromatography, as known in the art. Purified elute was then concentrated and redialyzed against water followed by lyophilization to obtain DCP6 test RSPP material (GC2596-26-E). The material was stored at -70ยฐ C. until needed for application testing, as described in the following Examples.
Example 6
Isoelectric Point and Cloud Point Determinations
[0197] In this Example, experiments performed to determine the isoelectric point and cloud point of SELP47K (Example 1) are described. The isoelectric point of SELP47K was determined by electrophoresing 5 mg/ml SELP47K solution using Isoelectric Focusing Electrophoresis (IEF) gel and Novex electrophoresis apparatus along with protein standards markers. Analysis of the gel revealed that SELP47K is a cationic protein with an isoelectric point of 10.4. Analysis of the IEF gels for isoelectric point determination of other RSPP examples reported in Table 2 were found to be within (+/-0.2) range.
[0198] The cloud point of SELP47K was determined by first preparing 0.1% and 1% solutions of polydispersed SELP 47K (Example 2) in three different buffers: 50 mM acetate pH 5; 50 mM Tris pH 7; 50 mM Tris pH 9. A 0.5 M NaCl salt solution was prepared in water and salt containing solutions were prepared using this stock solution. Samples were placed in quartz cuvettes and the cloud point determination was carried out using a Cary 300 spectrophotometer equipped with a thermal program and multiple cuvettes configuration. Turbidity measurements were made at 300 nm and the temperature was varied from 20-80ยฐ C.
[0199] It was determined that increasing the concentration of SELP47K in water lowered the cloud point of the protein polymer. A 0.05% solution of protein remains soluble in water even at 80ยฐ C. However, a 1% solution has a cloud point of 70ยฐ C. It was also determined that increasing pH from 5 to 9 also has a lowering effect on the cloud point of the protein polymer in very dilute solutions. At pH 5, the 0.1% solution remained soluble even at 80ยฐ C. At pH 9, the cloud point of 0.1% protein solution dropped down to 60ยฐ C. The 1% protein polymer solution showed similar cloud point of ห65ยฐ C. in the 5-9 pH range.
[0200] It was also determined that increasing the salt concentration also lowered the cloud point of the SELP47K protein polymer. In pH 5 buffer, 1% protein polymer solution has a cloud point of 75ยฐ C. However, the cloud point was 65ยฐ C. with 0.2 M salt and below 60ยฐ C. when the salt concentration was raised to 0.5 M. The presence of a high salt concentration in dilute protein solutions had more affect on the cloud point than that of a relatively less dilute solution of protein polymer. A 0.1% solution had a cloud point of 50ยฐ C. when 0.5 M NaCl was present in the solution irrespective of pH. A 1% solution with 0.5 M salt has cloud point of 55ยฐ C. irrespective of pH.
Example 7
Determination of the Glass Transition Temperature and Decomposition Temperature of SELP47K
[0201] In this Example, experiments performed to determine the glass transition temperature (Tg) and decomposition temperature of SELP47K. (Example 1) are described. Approximately 10 mg of SELP47K material was crimped in an aluminum (Al) pan, cooled to <-150ยฐ C., then heated to >200ยฐ C. at 10ยฐ C./min under a He (helium) atmosphere. The sample displayed an endothermic peak ranging from approximately 6 to 180ยฐ C. This initially appeared to be followed by another endothermic region and the analysis was stopped near 225ยฐ C. The sample was examined and had darkened significantly from the light golden starting color. Another sample of material was examined by TGA and found to have mass loss (-8%) from room temperature to approximately 200ยฐ C., confirming the broad endotherm observed with the first sample.
[0202] A strip of SELP47K was examined by using a dynamic mechanical analyzer (DMA) over the range of -150 to 200ยฐ C. Three tan delta peaks were observed at -109, 70, and 189ยฐ C. The peak at 189ยฐ C. coincided with a significant drop in modulus and a step change half-height in a differential scanning calorimeter (DSC) run in the same region initially was thought to be another endotherm suggesting the Tg of the material is approximately 189ยฐ C. Another DSC sample was run, pre-heating at 100ยฐ C. for 1 hour initially to remove volatiles. The mass loss endotherm was mostly removed, and a transition at 188ยฐ C. (half-height) remained. This run was stopped and the sample cooled and examined.
[0203] Based upon the results obtained, the material has a glass transition at approximately 189ยฐ C. Since there is mass loss on heating (water loss), this transition would be for the "film" material. Material degradation also appears to start near Tg. The decomposition temperature of this SELP47K was found to be 332ยฐ C.
Example 8
Tensile Mechanical Properties of SELP47K
[0204] In this Example, experiments performed to determine tensile mechanical properties of SELP47K (Example 1 and 2) are described. Samples of SELP47K were prepared by freeze-drying purified SELP47K solution. The subsequent powder was re-dissolved in water to a solids concentration of approximately 5, 10 or 20%. These solutions were then mixed with plasticizers to an effective concentration of 1-3%. Plasticizers included in this study were polyethylene glycol 200, glycerin, and triethanolamine. Eleven milliliters of these solutions were poured into a large (6 inch square) polyethylene weigh boats and the solutions were allowed to dry either in an oven at 37ยฐ C. or room temperature for 1 to 3 days. Subsequent films were successfully peeled from the weigh boat with a spatula without any damage to the polymer film.
[0205] The films were then cut into tensile specimens using a very sharp fabric cutting wheel. The specimens were approximately 6 cm long and 2.5 cm wide. The gauge length for these samples (length of sample between tensile testing clamps) was set to 3 cm and the thickness of the sample was measured at 5 points along the gauge length using a digital thickness coating instrument. The thickness of the samples was recorded as the average of the 5 points and varied from 46.4 to 188.2 microns. Both the sample width and sample thickness were entered into an Instron software program (Merlin) to enable it to calculate the tensile strength and tensile modulus.
[0206] The samples were loaded into the jaws of an INSTRONยฎ Model 5564 apparatus making sure the gauge length was always set to 3 cm and that the sample was not under any static load or stress. The 2,500 Newton (2.5 KN) load cell was used since these samples exhibited loads between 55 and 150 Newtons at break. The results of the stress-strain measurements can be seen for four of the samples in FIG. 1. The elongation % of SELP47K was determined to be 8.4 for 20% SELP47K solution film made at room temperature (RM). The tensile strength was determined to be 65.9 megapascals (MPa), and the tensile modulus was determined to be 1716.50 MPa. The results of plastisizer-SELP47K films reveal strong correlation between concentration and mechanical properties measured. For 3% PEG200 containing SELP47K, the films elongation % reached 541. The tensile strength (MPA) and tensile strain (%) for each protein polymer tested are provided in Table 3.
TABLE-US-00009 TABLE 3 a (Example 1 Material [GC2596-26-A]) Tensile Tensile Protein Polymer Strength (MPA) Strain (%) SELP47K (10% sol., 37 C.) 62.06 3.6 SELP47K (10% sol., RM) 62.98 3.5 SELP47K (20% sol., 37 C.) 74.14 8.6 SELP47K (20% sol., RM) 65.90 8.4 SELP47K (05% sol., 37 C.) 19.83 2.6 SELP47K (05% sol., RM) 21.43 2.4 SELP47K (20% sol., RM) + 27.65 180.5 2% Glyerol SELP47K (20% sol., RM) + 42.25 541.7 2% PEG 200 SELP47K (20% sol., RM) + 67.84 7.9 1% PEG 200 SELP47K (20% sol., RM) + 49.65 87 3% TEA SELP47K (20% sol., RM) + 54.96 31 1% TEA Nylon 6 50.56 50.6 Polystyrene 42.66 1.8 Low Density Polyethylene 49.98 375.4 b (Example 2 Material [GC2596-26-B]) Tensile Tensile Protein Polymer Strength (MPA) strain (%) SELP47K (10% sol., 37 C.) 39.34 6.98 SELP47K (10% sol, 37 C.) + 25.12 15.75 1% glycerol
Example 9
SELP47K Film
[0207] This Example describes the casting of a SELP47K (Example 1) films. First, a thin film was cast, starting with a 10% solution of SELP47K in water was prepared. The spin coater used for making thin film was from Special Coating Systems Inc., model number P6708-D (Indianapolis, USA). The DV-1000 program was used for spinning the film. SELP47K protein solution was poured over a 316 stainless steel plate that was placed on the spinner. The spinner was rotated at 2000 rpm for 30 seconds followed by rotation at 1000 rpm for 90 seconds. The thin film was allowed to dry for an hour and the thickness of the film was measured using a magnetometer.
[0208] The average thickness of the SELP47K thin film was between 2-5 ฮผm. A SELP47K (Example 1) thick film was cast in the following manner. A 10% solution of SELP47K was prepared in deionized water (DI). This solution was poured to 1 mm in height into a plastic container and was allowed to set at 37ยฐ C. or room temperature for several hours leading to the formation of films. These films averaged between about 50-200 ฮผm in thickness.
[0209] A SELP47K (Example 1) clear thick film was cast in the following manner. A 5% solution of SELP47K was prepared in DI water. This solution was poured to 1 mm thickness on a sheet of Saran Wrapยฎ kept in a shallow plastic container. This container was maintained at room temperature overnight leading to the formation of a clear thick film of SELP47K. SELP47K film made though this method yielded excellent optical transparency. No X-ray diffraction pattern was obtained from this material, and, thus, the material of this film was amorphous in nature. A 1% water solution of SELP47K when analyzed by circular dichroism spectroscopy, predicted 50% random coil and 50% beta sheet structure.
Example 10
Microscopy of SELP47K
[0210] This Example describes microscopic studies of SELP47K (Example 1 and 2 material). In these experiments, the morphology of Silk Elastin Protein (SELP47K) was characterized using various microscopy techniques including: 1) optical microscopy; 2) Field Emission Scanning Electron Microscopy (FE-SEM); and 3) Atomic Force Microscopy (AFM).
[0211] Four concentrations of the SELP material (13.5%, 1.35%, 0.135% and 0.0135%) were prepared in distilled water and spin-coated on to the surface of a plasma-treated wafer (hydrophilic surface) for examination. FIGS. 2 and 3 provide photomicrographs of SELP 47-K film showing self assembly into nanofilaments.
[0212] AFM and SEM examination indicated that there was a coating over the entire surface of the wafer. The coating appeared to be comprised of densely packed intertwining strands. Microscopy results indicated that the SELP47K coating, when concentrated, is comprised of densely packed, long, intertwined strands. Short, single/double strands of material were found in areas where the coating appeared to be less concentrated. Individual strands of material could be seen in the less concentrated, gray areas on the wafer. SEM analysis of the films provide the basis of water-solubility of SELP47K films. A SELP47K film made out of 10% SELP47K solution gave a water-soluble film whereas a 20% solution film was water insoluble. In 10% SELP47K solution film, micellar droplet structures appeared to be responsible for its water solubility whereas 20% SELP47K solution film did not have the micellar droplet structures in the film.
[0213] These strands ranged from 100 nm to 1 ฮผm in length and 20-45 nm in diameter. These experiments confirm that SELP47K can be spun into a film and this film when studied using microscopic techniques reveal that film is composed in a non-woven web of filaments. The film is composed of nano filaments. Nano fibers of this protein can be thus utilized in various applications, such as personal care products, due to their strength, advantage of very large surface to mass ratio and protein based targeting characteristics.
Example 11
Preparation of Rinse-Off Conditioners
[0214] In this Example, preparation of rinse-off conditioners is described. The compositions for the conditioners are set forth in Table 4.
TABLE-US-00010 TABLE 4 Rinse-Off Conditioner Components Composition Composition Composition (Weight %) (Weight %) (Weight %) Ingredient A B C Deionized Water q.s. to 100 q.s. to 100 q.s. to 100 Hydroxyethylcellulose1 1.5 1.5 1.5 Cetearyl Alcohol2 1.0 1.0 1.0 PEG-100 Stearate & 1.0 1.0 1.0 Glyceryl Stearate3 Hydrolyzed Silk Protein4 5.0 -- -- Hydrolyzed Elastin -- 4.0 -- Protein5 Pure Polydispersed -- -- 1.0 Silk-Elastin Protein6 DMDM Hydantoin7 0.2 0.2 0.2 1NATROSOL ยฎ 250 MR (Hercules) 2LANETTE O ยฎ (Cognis Corp.) 3ARLACEL ยฎ 165 (Uniqema) 4Crosilk 10,000 (Croda) Equivalent weight % of protein in conditioner A is 1.0%. 5Crolastin (Croda). Equivalent weight % of protein in conditioner B is 1.0%. 6Silk Elastin protein prepared in accordance with Example 2. Equivalent weight % of protein in conditioner C is 1.0%. 7GLYDANT ยฎ (Lonza)
[0215] In order to prepare the compositions, deionized water was added to a mixing vessel and heated to 75ยฐ C. With moderate agitation, the hydroxyethyl cellulose was dispersed until fully dissolved. The heat was decreased to 60ยฐ C. and cetearyl alcohol and PEG-100 stearate and glyceryl stearate were added. The heat was then decreased to 40ยฐ C. and then the protein for either the A, B, or C conditioner was added. The conditioner was mixed for 5-10 minutes and then DMDM hydantoin was added. The water loss was compensated for and the formulation was mixed for an additional 5 minutes. The final pH of the conditioner formulations was approximately 6-7.
[0216] Slightly bleached European human hair from International Hair Importer and Products Inc. was used for testing these conditioners. A master hand of hair about eight inches in length was subdivided into a series of individual hair tresses. Each tress weighed about 2.5 grams. A1/2 inch portion of the root end of the hair was trimmed and glued to a 2''ร2'' plastic tab using DUCO CEMENTยฎ. The final weight of each tress was approximately 2.0 g. The cement was allowed to dry, and the hair tress was combed and trimmed to a length so that six inches of hair was extended below the bottom of the plastic tab. A hole was punched in middle of tab 1/4'' from the top. Each tress was rinsed for 15 seconds under 40ยฐ C. tap water. Using a pipette, 1.0 g of a 9% sodium lauryl sulfate (active) solution was applied and lathered through the tress for 30 seconds. The tress was rinsed for 30 seconds under running water. Excess water was removed from the tress by passing the tress between the index and middle fingers. The tresses were placed on a tray covered with paper towels and dried overnight. Each tress was hand combed three times with the narrow teeth of an ACEยฎ comb and evaluated using the INSTRONยฎ "WET" and the INSTRONยฎ "DRY" COMBING procedures.
[0217] For tests involving the rinse-off conditioner, the hair tress was rinsed with tap water for 15 seconds at 40ยฐ C. The test conditioner was applied to the tress in the amount of 0.8 g and the tress was stroked for 30 seconds. The tress was rinsed for 30 seconds under tap water at 40ยฐ C. The excess water was removed by pulling the tress through the index and middle fingers. The tresses were allowed to dry separately on a paper towel, overnight at room temperature. The tresses were combed once before performing the INSTRONยฎ study.
[0218] INSTRONยฎ COMBING is an industry recognized test for determining hair conditioning by the ease of wet combing and the ease of dry combing. The test employs an INSTRONยฎ strain gauge, which is equipped to measure the force required to comb the hair. Conditioning performance is based on the ability of a particular hair treatment formulation such as a shampoo or a hair conditioner to reduce the force required to comb the hair with the INSTRONยฎ strain gauge. The force is reported as Average Combing Load (ACL). The lower ACL value, the better the conditioning effect imparted by the formulation being tested. Typically, ACL baselines are initially established with "untreated" tresses that have only been washed with the sodium lauryl sulfate solution. The effectiveness of a treatment can be expressed as the ACL of the treated tress or the % Reduction in ACL which is calculated by ((untreated hair ACL-treated hair ACL)/untreated hair ACL)*100.
[0219] According to the INSTRONยฎ WET COMBING method, the hair was first wet by dipping it in distilled water and then the hair was detangled by combing the tress three times. The tress was then retangled by dipping in distilled water three times. The excess water was removed by passing the tress through index and middle fingers twice. The tress was then placed on the hanger and INSTRON combed. The "retangle" and "INSTRONยฎ combing" steps were repeated until all data points were collected. An average combing force of three tresses was measured for each treatment. The results of the INSTRONยฎ WET COMBING test conducted with the conditioners of the present invention are shown below in Table 5. Letters in the % ACL Reduction column are used to indicate that the product is superior to other designated products at a 90% confidence level. The results show that the performance of the conditioner containing the polydispersed silk elastin compound of the present invention provided a small reduction in dry combing forces compared to the conditioners that contained the hydrolyzed silk and elastin proteins. The performance of these two conditioners actually showed an increase in combing forces compared to the untreated tress.
TABLE-US-00011 TABLE 5 INSTRON ยฎ WET COMBING Conditioner Tested ACL Reduction (%)a A -22 B -32 C 3 (A, B) aLetter designations indicate that the conditioner was significantly different from the specified conditioners at a 90% confidence level
[0220] According to the INSTRONยฎ DRY COMBING method, the hair was detangled by combing the tress 3 times. Then, the hair was retangled by swirling the tress clockwise 3 times and counter clockwise 3 times. The tress was then placed on the hanger and INSTRONยฎ combed. The "retangle" and "INSTRONยฎ combing" steps were repeated until all data points were collected. An average combing force of three tresses was measured for each treatment. The results of the INSTRONยฎ DRY COMBING test conducted with the conditioners are shown below in Table 6. The results show that the performance of the silk elastin composition of the present invention provided a larger reduction in dry combing forces compared to the conditioners that contained hydrolyzed silk protein and hydrolyzed elastin proteins, respectively.
TABLE-US-00012 TABLE 6 INSTRON ยฎ DRY COMBING Conditioner Tested ACL Reduction (%)a A 20 (B) B -14 C 33 (A, B) aLetter designations indicate that the conditioner was significantly different from the specified conditioners at a 90% confidence level.
[0221] Additional rinse-off conditioners were also prepared. The compositions of this second set of rinse-off conditioners are set forth in Table 7.
TABLE-US-00013 TABLE 7 Second Set of Rinse-Off Conditioners Composition Composition Composition (Weight %) (Weight %) (Weight %) Ingredient A B C Deionized Water q.s. to 100 q.s. to 100 q.s. to 100 Hydroxyethylcellulose 1.5 1.5 1.5 Cetearyl Alcohol 1.0 1.0 1.0 PEG-100 Stearate & 1.0 1.0 1.0 Glyceryl Stearate Pure Polydispersed 0.01 -- -- Silk-Elastin Protein1 Pure Polydispersed -- 0.1 -- Silk-Elastin Protein1 Pure Polydispersed -- -- 1.0 Silk-Elastin Protein1 DMDM Hydantoin 0.2 0.2 0.2 1Silk-elastin protein prepared in accordance with Example 2.
[0222] INSTRONยฎ wet and dry combing tests were performed using this second set of rinse-off conditioners, as described above. The INSTRONยฎ combing results in Tables 8 and 9 show the effect of concentration for the pure, polydispersed silk elastin protein compound. Table 9 shows that the performance of the protein material provided a reduction in dry combing force at a concentration as low as 0.1% in the rinse-off conditioner formulation, thereby improving the conditioning properties of the hair.
TABLE-US-00014 TABLE 8 INSTRON ยฎ WET COMBING Conditioner Tested ACL Reduction (%)a A -25 B -2 C 3
TABLE-US-00015 TABLE 9 INSTRON ยฎ DRY COMBING Conditioner Tested ACL Reduction (%)a A -5 B 11 C 33 (A) aLetter designation indicates that the conditioner is significantly different to the specified conditioner at a 95% confidence level.
[0223] An additional set of rinse-off conditioners were prepared. The compositions of this third set of rinse-off conditioners are set forth in Table 10.
TABLE-US-00016 TABLE 10 Third Set of Rinse-Off Conditioners Composition Composition Composition (Weight %) (Weight %) (Weight %) Ingredient A B C Deionized Water q.s. to 100 q.s. to 100 q.s. to 100 Hydroxyethylcellulose 1.5 1.5 1.5 Cetearyl Alcohol 1.0 1.0 1.0 PEG-100 Stearate & 1.0 1.0 1.0 Glyceryl Stearate Pure Monodisperse Silk 0.01 -- -- Elastin Protein1 Pure Monodisperse Silk -- 0.1 -- Elastin Protein1 Pure Monodisperse Silk -- -- 1.0 Elastin Protein1 DMDM Hydantoin 0.2 0.2 0.2 1Silk Elasin protein prepared in accordance with Example 1.
[0224] INSTRONยฎ wet and dry combing tests were performed in conjunction with the procedures described above. The INSTRONยฎ combing results in Tables 11 and 12 show the effect of concentration for the pure, monodispersed silk elastin protein compound. Table 12 shows that the performance of the protein material provided a small reduction in dry combing force at all concentrations in the rinse-off conditioner formulation, thereby improving the conditioning properties of the hair.
TABLE-US-00017 TABLE 11 INSTRON ยฎ WET COMBING Conditioner Tested ACL Reduction (%)a A -17 B -4 C -49 aLetter designation indicates that the conditioner was significantly different from the specified conditioner at a 95% confidence level.
TABLE-US-00018 TABLE 12 INSTRON ยฎ DRY COMBING Conditioner Tested ACL Reduction (%) A 10 B 9 C 5
Example 12
Efficacy Studies of SELP47K Applied to Skin
[0225] This Example describes experiments conducted in an efficacy study to measure changes in the biomechanical properties of skin and the ability of a single application of SELP to diminish the visual effects of aging on skin.
[0226] Eleven impaneled subjects (age 35-70 years) showing clear signs of facial skin aging were provided with a non-moisturizing soap, (AVEENOยฎ), one week prior to the study and were instructed to use the provided soap to wash the face and were required to refrain from excessive UV exposure. After a seven-day conditioning phase, subjects were acclimated to the ambient temperature and humidity for thirty minutes. One side of the face of each subject was designated as the measurement side by random selection by computer. After 30-minute acclimation period, baseline digital photographs were taken, and instrumental measurements, skin replica samples and visual evaluations were made. Photographs were taken, with a Canfield Scientific Camera, of the full face, the periorbital area, and the temporal side of eye. Chromameter (Model CR300, Minolta) measurements were taken from the periorbital area directly under the eye (Chardon et al., Int. J. Cosmet. Sci., 13:191-208
[1991]). Silflo replicas (CuDerm Corporation, Dallas, Tx) were made from the periorbital area adjacent to the temporal side of the eye (Grove et al., J. Am. Acad. Dermatol., 21:631-637
[1998]; and Sun et al 1997 IFSCC Conference, Mexico). Cutometer (SEM575, Courage and Khazaka, Germany) measurements were taken at the periorbital area at the upper portion of cheekbone. All measurements were performed in triplicate. An assessment of the relative depth and frequency of facial lines was performed by the method of Packman and Gans (Packman and Gans, J. Soc. Cosmet. Chem., 29:79-90
[1978]).
[0227] After baseline control data were collected, 5% SELP47K aqueous solution was applied to the face of each subject. A few drops of the SELP47K solution was dispensed onto the fingertips and smoothed into the skin of the face. Subjects were retained in the lab for 30 minutes after application of the SELP47K aqueous solution, at which point a second set of measurements were made.
[0228] Image analysis of Silflo replicas was done. Specifically, the fine line factors, which approximate the number and/or length of facial lines in the 10 evaluated bands of skin replica, were measured. At 30 minutes after application of SELP47K, fine line factors decreased by a statistically significant 13% (p=0.05). The statistical comparisons of the fine lines factors are summarized in Table 13.
TABLE-US-00019 TABLE 13 Statistical Comparisons of Fine Lines Factors Fine Line Factors Baseline 30 Minutes Post Mean 284 246 Variance 1584 2538 Observations 11 11 Pearson Correlation 0.16 Hypothesized Mean 0 df 10 t Stat 2.18 P(T <= t) one-tail 0.03 t Critical one-tail 1.81 P(T <= t) two-tail 0.05 t Critical two-tail 2.23
[0229] Evaluation of superficial facial lines was performed by an expert evaluator using the scoring method described by Packman and Gans (supra). A 7% decrease in the superficial facial line score was assessed at the 30-minute post-treatment interval compared to baseline, which was statistically significant at the 91% confidence level (p=0.09). The statistical comparisons of these measured values are summarized in Table 14.
TABLE-US-00020 TABLE 14 Statistical Analysis of Superficial Facial Line Scores Superficial Facial Line Score Baseline 30 Minutes Post Mean 40 37 Variance 640 660 Observations 11 11 Pearson Correlation 0.98 Hypothesized Mean 0 df 10 t Stat 1.90 P(T <= t) one-tail 0.04 t Critical one-tail 1.81 P(T <= t) two-tail 0.09 t Critical two-tail 2.23
[0230] Chromometer values for L*, a*, and b* were read and total color value, E, was calculated for each subject. The average a* value (which is directly related to the redness of the skin) decreased with statistical significance by 6% relative to baseline (p=0.024) as shown in Table 15.
TABLE-US-00021 TABLE 15 Chromameter Scores Chromameter a* Score Baseline 30 Minutes Post Mean 11.05 10.39 Variance 2.08 2.74 Observations 11 11 Pearson Correlation 0.86 Hypothesized Mean 0 df 10 t Stat 2.66 P(T <= t) one-tail 1.20 E-02 t Critical one-tail 1.81 P(T <= t) two-tail 2.39 E-02 t Critical two-tail 2.23
[0231] Cutometer results showed an increase in Uv (a measure of viscoelasticity and delayed distention of the skin), indicating an increase in skin softness, and was assessed at a statistical confidence level of 91% (p=0.09) as shown in Table 16.
TABLE-US-00022 TABLE 16 Cutometer Scores Cutometer Uv Score Baseline 30 Minutes Post Mean 0.09 0.106 Variance 0.000 0.001 Observations 11 11 Pearson Correlation 0.06 Hypothesized Mean 0 df 10 t Stat -1.88 P(T <= t) one-tail 0.04 t Critical one-tail 1.81 P(T <= t) two-tail 0.09 t Critical two-tail 2.23
[0232] In summary, as observed in the above experiments, the foremost effects of a single treatment with 5% aqueous solution of SELP47 K were reductions in both the appearance and the measured number and/or length of fine lines in the periorbital area of the face as well as an indication of improved skin softness and the evenness of tone.
Example 13
Personal Care Compositions
[0233] This Example provides various personal care compositions comprising any of the compounds
TABLE-US-00023 BODY WASH BODY WASH RAW MATERIAL pH 6.5 PH 7 PH 8 (INCI Designation) Amount Amount Amount Deionized water QS QS QS Sodium Laureth Sulfate 12 15 8 Cocamidopropyl Betaine 8 10 15 DecylAPG Glucoside 0 2 1 (Plantacare 2000 1) Polyquaternium-10 0.25 0 0 (JR30M) Polyquaternium-7 0 0 0.7 (Mackam 55) Preservative, fragrance, QS QS QS color
TABLE-US-00024 MOISTURIZING BODYWASH RAW MATERIAL pH = 7 (INCI Designation) Amount Deionized Water QS Glycerin 4.0 PEG-6 Caprylic/Capric Glycerides 4.0 Palm Kernel Fatty acids 3.0 Sodium Laureth-3 Sulfate 45.0 Cocamide MEA 3.0 Sodium Lauroamphoacetate 25.0 Soyabean Oil 10.0 Polyquaternium-10 (JR30M) 0.70 Preservative, fragrance, color QS Repeat Sequence Protein Polymer 1000 ppm Repeat Sequence Protein 250 ppm 500 ppm 1000 ppm Polymer
TABLE-US-00025 BODY LOTION RAW MATERIAL pH 7 pH 7 pH 7.5 pH 7 (INCI Designation) Amount Amount Amount Amount Deionized Water QS QS QS QS Glycerine 8 8 10 12 Isohexadecane 3 3 3 6 Niacinamide 0 3 5 6 Isopropyl Isostearate 3 3 3 3 Polyacrylamide (and), 3 3 3 3 Isoparaffin, (and) Laureth-7 (Sepigel 305 2) Petrolatum 4 4 4 2 Nylon 12 2 2 2.5 2.5 Dimethicone (DC1403 4) 2 2 2.5 2.5 Sucrose 1.5 1.5 1.5 1.5 Polycottonseed Oil Stearyl Alcohol 97% 1 1 1 1 D Panthenol 1 1 1 1 DL-alphaTocophero 1 1 1 1 Acetate Cetyl Alcohol 95% 0.5 0.5 0.5 1 Behenyl Alcohol 1 1 1 0.5 Cetearyl Alcohol 0.4 0.4 0.5 0.5 (and) Cetearyl GlucosidePL 68/50 Stearic Acid 0.15 0.15 0.15 0.15 PEG-100-Stearate 0.15 0.15 0.15 0.15 (MYRJ 59 1) Preservative, QS QS QS QS fragrance, color Repeat sequence 250 ppm 500 ppm 750 ppm 1000 ppm protein polymers
TABLE-US-00026 LEAVE-ON HAIR CONDITIONER RAW MATERIAL (INCI Designation) Amount Deionized Water QS Isostearamidopropyl Morpholine Lactate 6.0 Hydroxyethylcellulose 1.0 Preservative, fragrance, color QS Repeat sequence protein polymers 1000 ppm
TABLE-US-00027 CONDITIONING SHAMPOO RAW MATERIAL (INCI Designation) Amount Deionized Water QS Sodium Laureth Sulfate 30% 27.0 Cocamidopropyl Betaine 3.7 Coco-Glucoside (and) Glyceryl Oleate 5.0 Coco-Glucoside (and) Glycol Distearate (and) Glycerine 3.0 Guar Hydroxypropyl Trimonium Chloride 0.1 Laureth-2 1.55 Fragrance, preservative, color QS Repeat sequence protein polymer 1000 ppm
TABLE-US-00028 CREAM RINSE RAW MATERIAL pH 4 (INCI Designation) Amount Deionized Water QS Behentrimonium Chloride 2.0 Trilaureth-4 Phosphate 1.5 Cetyl alcohol 2.0 Citric acid QS Preservative, fragrance, color QS Repeat sequence protein polymer 1000 ppm
TABLE-US-00029 NOURISHING HAIR CONDITIONER/TREATMENT RAW MATERIAL pH 6 (INCI Designation) Amount Deionized Water QS Behentrimonium Methosulfate (and) Cetyl Alcohol 4.0 Wheat germ oil 1.0 Cetyl alcohol 0.5 Propylene glycol 5.0 PEG-60 Lanolin 1.0 Panthenol 2.0 Lupin amino acids 1.0 Cocodimonium Hydroxypropyl Hydrolyzed Wheat Protein 1.0 Fragrance, preservative, color QS Repeat sequence protein polymer 1000 ppm
TABLE-US-00030 ANTI-DANDRUFF SHAMPOO RAW MATERIAL (INCI Designation) Amount Deionized Water QS Magnesium Aluminum Silicate 1.0 Hydroxypropyl Methylcellulose 0.8 Sodium Olefin Sulfate 40% 35.0 Lauramide DEA 4.0 Soyamide DEA 1.0 Quaternium-70 Hydrolyzed Collagen 2.0 Zinc Pyrithione 40% 4.0 Fragrance, preservative, color QS Repeat sequence protein polymer 1000 ppm
TABLE-US-00031 ULTRA-HIGH MOISTURIZING FACIAL CREAM/LOTION EMULSION pH 7 pH 7 RAW MATERIAL (INCI Designation) Amount Amount Deionized water QS QS Glycerin 12 5 PEG 4006 0 10 Niacinamide 5 7 Isohexadecane 5 5 Dimethicone (DC14033) 3 2 Polyacrylamide (and), Isoparaffin 3 3 (and), Laureth-7 (Sepigel 3051) Isopropyl Isostearate 2 2 Polymethylsilsesquioxane 2 2 Cetyl Alcohol 95% 1 1 Sucrose polycottonseed oil 1 1 D-Panthenol 1 1 Vitamin E (Tocopherol Acetate) 1 1 Stearyl Alcohol 95% 0.5 0.5 Cetearyl Glucoside 0.5 0.5 Titanium dioxide 0.3 0.3 Stearic Acid 0.15 0.15 PEG-100-Stearate (Myrj 594) 0.15 0.15 Preservative, fragrance, color QS QS Repeat sequence protein polymer 250 ppm 1500 ppm MOISTURIZING CREAM pH 7 pH 7 pH 7.5 RAW MATERIAL (INCI Designation) Amount Amount Amount Deionized water QS QS QS Glycerine 3 5 10 Petrolatum 3 3 0 Cetyl Alcohol 95% 1.5 1.5 1 Dimethicone Copolyol (DC 2 2 2 3225C4) Isopropyl Palmitate 1 1 0.5 Carbopolmer 954 (Noveon) 0.7 0.7 0.7 Dimethicone (DC 200/350cs4) 1 1 1 Stearyl Alcohol 97% 0.5 0.5 1 MOISTURIZING CREAM pH 7 pH 7 pH 7.5 Stearic acid 0.1 0.1 0.1 PEG-100-stearate (MYRJ 591) 0.1 0.1 0.1 Titanium Dioxide 0.3 0.3 0.3 Preservative, color, fragrance QS QS QS Repeat sequence protein polymer 50 ppm 250 ppm 1000 ppm
TABLE-US-00032 FACIAL CLEANSING EMULSION RAW MATERIAL (INCI Designation) Amount Water 69.05 Disodium EDTA 0.1 Glyceryl polymethacrylate (and) Propylene glycol 1.0 Glycerin 2.0 Xanthan gum 0.5 Hydroxyethyl cellulose 0.5 Tridecyl neopentanoate 4.0 Isocetyl stearate 6.0 Octyl palmitate 8.0 Glyceryl dilaurate 4.0 PEG-20 stearate 2.0 Glyceryl stearate (and) Laureth-23 2.0 Lauryl pyrrolidone 0.5 Chamomile extract 0.2 Aloe vera (200x) 0.05 Fragrance, preservative QS Repeat sequence protein polymer 0.1
TABLE-US-00033 SURFACTANT-BASED FACIAL CLEANSER RAW MATERIAL (INCI Designation) Amount Water 62.55 Acrylates/Steareth-20 methacrylate copolymer 3.3 Disodium EDTA 0.05 Glycerin 2.0 Glyceryl polymethacrylate (and) Propylene glycol (and) 0.5 PVM/MA copolymer Sodium laureth sulfate (30%) 17.5 Cetearyl alcohol 1.0 Shea butter 1.0 Disodium oleamido PEG-2 sulfosuccinate 5.0 Cocoamidopropyl Betaine 3.0 Sodium lauroyl sarcosinate 1.0 PEG-7 glyceryl cocoate 1.0 Isodecyl oleate 1.5 Peppermint extract 0.25 Eucalyptus extract 0.25 Fragrance, preservative, color, pH adjust QS Repeat sequence protein polymer 0.1
TABLE-US-00034 FACIAL EXFOLIATING GEL RAW MATERIAL (INCI Designation) Amount Water 64.39 Disodium EDTA 0.05 Aloe vera (200x) 0.01 Benzophenone-4 0.25 Propylene glycol 1.0 Acrylates/C10-30 alkyl acrylate crosspolymer (2%) 20.0 Glyceryl polymethacrylate (and) Propylene glycol 10.0 Glyceryl polymethacrylate (and) Propylene glycol 1.0 (and) PVM/MA copolymer Hydrogenated jojoba oil 1.5 Fragrance, preservative, color, pH adjust QS Repeat sequence protein polymer 0.05
TABLE-US-00035 FACIAL TONER RAW MATERIAL (INCI Designation) Amount Water 93.99 Disodium EDTA 0.1 Butylene glycol 2.0 Aloe vera (200x) 0.1 Allantoin 0.1 Benzophenone-4 0.5 Witch hazel extract 0.3 Propylene glycol (and) Euphrasia extract (and) 0.01 Golden seal root extract (and) Green tea extract PEG-40 hydrogenated castor oil 0.5 Quaternium-22 0.5 Sandlewood oil 0.02 Fragrance, preservative, color, pH adjust QS Repeat sequence protein polymer 0.05
TABLE-US-00036 EXFOLIATING CREAM RAW MATERIAL (INCI Designation) Amount Water 68.80 Disodium EDTA 0.1 PVM/MA decadiene crosspolymer 1.0 Butylene glycol 3.0 PEG-20 stearate 1.0 Glyceryl stearate (and) Laureth-23 2.0 Diisopropyl adipate 2.0 Isodecyl oleate 2.0 Isocetyl stearoyl stearate 5.0 Myristyl myristate 1.0 Glyceryl dilaurate 2.0 Sodium hydroxide, 10% 2.6 Glyceryl polymethacrylate (and) Propylene glycol 5.0 Glyceryl polymethacrylate (and) Propylene glycol 0.5 (and) PVM/MA copolymer Hydrogenated jojoba oil 3.0 Fragrance, preservative, color, pH adjust QS Repeat sequence protein polymer 0.05
TABLE-US-00037 FACIAL MASK RAW MATERIAL (INCI Designation) Amount Water 76.4 Disodium EDTA 0.1 Bentonite 12.5 Potassium C12-13 Alkyl Phosphate 5.0 Propylene glycol 4.0 Sodium Coco PG-Dimonium Chloride Phosphate 1.0 Fragrance, preservative, color, pH adjust QS Repeat sequence protein polymer 0.05
TABLE-US-00038 AFTER SHAVE BALM RAW MATERIAL (INCI Designation) Amount Water 82.12 Disodium EDTA 0.1 Acrylate copolymer 2.0 Acrylate/Stareth-20 methacrylate copolymer 1.0 Propylene glycol 3.0 Sodium hydroxide (10%) 1.28 Glyceryl stearate (and) Cetyl alcohol (and) Stearyl alcohol 3.5 (and) Behenyl alcohol (and) Palmitic acid (and) Stearic acid (and) Hydroxyethyl cetearamidopropyldimonium chloride Isocetyl stearate 1.0 C12-15 alkyl lactate 1.5 Octyldodecyl stearate 3.0 Glyceryl polymethacrylate (and) Propylene glycol (and) 1.0 PVM/MA copolymer Polyquaternium-11 0.5 Fragrance, preservative, color, pH adjust QS Repeat sequence protein polymer 0.1
TABLE-US-00039 EYE GEL RAW MATERIAL (INCI Designation) Amount Water 89.14 VP/Acrylates/Lauryl methacrylate copolymer 0.5 Glycerin 5.0 Aminomethyl propanol 0.3 Aloe vera (200x) 0.05 Benzophenone-4 0.1 Glyceryl polymethacrylate (and) Propylene glycol (and) 0.2 PVM/MA copolymer Butylene glycol (and) Water (and) Witch hazel extract 0.5 Butylene glycol (and) Water (and) Cucumber extract 0.3 PEG-40 hydrogenated castor oil 0.01 Acrylates/Beheneth-25 methacrylate copolymer 2.4 Fragrance, preservative, color, pH adjust QS Repeat sequence protein polymer 1.0
TABLE-US-00040 HIGH MELTING POINT LIPSTICK RAW MATERIAL (INCI Designation) Amount Ozokerite wax 5.0 Candelilla wax 11.0 Octyl dodecanol 26.0 C30-45 alkyl methicone 5.0 Cyclomethicone 4.8 Petrolatum 3.0 Lanolin oil 9.0 Avocado oil 2.0 Oleyl alcohol 8.0 Pigment/cyclomethicone 25.0 Fragrance, preservative QS Repeat sequence protein polymer 1.0
TABLE-US-00041 LIPSTICK RAW MATERIAL (INCI Designation) Amount Candelilla wax 9.1 Isopropyl myristate 9.6 Lanolin 5.0 Beeswax 4.0 Paraffin (130/135) 2.0 Ozokerite wax 2.5 Castor oil 53.7 Carnauba wax 1.5 Pigments 7.5 Mineral oil 4.0 Fragrance, preservative QS Repeat sequence protein polymer 1.0
TABLE-US-00042 LIP GLOSS RAW MATERIAL (INCI Designation) Amount Bis-diglyceryl polyacyladipate-1 43.5 Bis-diglyceryl polyacyladipate-2 10 Glycerol ricinoleate 10 Polyisobutene 1000 13 Lanolin wax 10 Candelilla wax 2.5 Mica (and) titanium dioxide 3 d-Panthenol 5 Fragrance, preservative, color QS Repeat sequence protein polymer 1.0
TABLE-US-00043 LIP GLOSS WITH SUNSCREEN RAW MATERIAL (INCI Designation) Amount Triisostearyl Citrate 58.4 Candelilla wax 8.0 Myristyl lactate 7.5 Microcrystalline wax 5.0 Carnauba wax 2.0 Diisopropyl dimmer dilinoleate 10.0 Mica (and) Bismuth oxychloride (and) Carmine 6.0 Zinc oxide (microfine) 2.0 Fragrance, preservative QS Repeat sequence protein polymer 1.0
TABLE-US-00044 LIP BALM RAW MATERIAL (INCI Designation) Amount Petrolatum 47.3 Isopropyl lanolate 6.0 Ozokerite wax 16.5 Candelilla wax 4.5 Diisopropyl dilinoleate 25.0 Retinyl palmitate 0.5 Tocopherol acetate 0.2 Fragrance, preservative QS Repeat sequence protein polymer 1.0
TABLE-US-00045 WATERPROOF MASCARA RAW MATERIAL (INCI Designation) Amount Water 49.45 Propylene glycol 3.0 Triethanolamine (99%) 3.1 Acrylates/Octylacrylamine Copolymer 5.0 Diisostearoyl trimethylolpropane siloxy silicate 5.0 Candelilla wax 4.5 Beeswax 5.5 Ozokerite wax 2.0 Carnauba wax 1.0 Cetyl alcohol 3.0 Stearic acid 5.0 Iron oxides 11.0 Fragrance, preservative QS Repeat sequence protein polymer 2.0
TABLE-US-00046 ANHYDROUS WATERPROOF MASCARA RAW MATERIAL (INCI Designation) Amount C9-11 Isoparaffin 30.95 Polyethylene 11.0 Candelilla wax 4.5 Hydroxylated lanolin 0.25 Pentaerythrityl rosinate 2.0 Zinc stearate 1.0 Silica silylate 1.0 Petroleum distillates (and) Quaternium-18 hectorite 35.0 (and) Propylene Carbonate Iron oxides 12.0 Fragrance, preservative QS Repeat sequence protein polymer 2.0
TABLE-US-00047 WATER-BASED MASCARA RAW MATERIAL (INCI Designation) Amount Water 43.32 Polyvinyl pyrrolidone (K30) 2.0 Hydroxyethyl cellulose 1.0 Triethanolamine (99%) 2.0 Disodium EDTA 0.1 Iron Oxides 10.0 Stearic acid 4.5 Glyceryl monostearate 2.0 Beeswax 7.0 Carnauba wax 4.5 Hydroxylated lanolin 1.0 Acrylates copolymer 20.0 Fragrance, preservative QS Repeat sequence protein polymer 2.0
TABLE-US-00048 LIQUID EYELINER RAW MATERIAL (INCI Designation) Amount Water 50-70 Gellant 0.5-1.5 Wetting agent(s) 1-3 Polyol 4-8 Colorants 10-20 Alcohol 5-10 Film former 3-8 Fragrance, preservative QS Repeat sequence protein polymer 2.0
TABLE-US-00049 NAIL ENAMEL RAW MATERIAL (INCI Designation) Amount Solvent(s) 40-70 Resin(s) 10-20 Plasticizer 3-12 Gellant 0-2 Colorants 0-3 Fragrance, preservative QS Repeat sequence protein polymer 2.0
TABLE-US-00050 CUTICLE TREATMENT RAW MATERIAL (INCI Designation) Amount Petrolatum 34.8 Beeswax 7.2 Ozokerite wax 4.3 Candelilla wax 4.0 Cocoa butter 1.0 Shea butter 1.0 Glyceryl dilaurate 8.0 Ethylhexyl palmitate 20.0 C12-15 alkyl lactate 6.0 PVP/Eicosene copolymer 3.5 Diisopropyl adipate 2.0 Octinoxate 7.5 Retinyl palmitate 0.1 Tocopherol acetate 0.1 Fragrance, preservative, color, pH adjust QS Repeat sequence protein polymer 0.5
TABLE-US-00051 PRESSED POWDER FORMULATIONS Loose Pressed Founda- Eye Powder Powder tion Blush Shadow Fillers 70-95 40-90 40-80 40-80 40-80 (eg. talc, mica, seracite) Compression aids .sup. 0-2.5 3-5 2-5 2-7 2-10 (e.g., metallic soaps, waxes) Texture enhancers 10-40 5-40 10-40 10-40 0-30 Colorants 2-10 2-10 5-20 2-10 1-40 (e.g., iron oxides, organic colors) Pearls 0-20 0-10 0-5 0-20 0-60 (e.g. titanated mica, bismuth oxychloride) Wet binder 0-3 2-5 2-5 3-10 3-15 (e.g., Octyldodecyl stearoyl stearate, di-PPG3 myristyl ether adipate, isocetyl stearate, cetyl dimethicone) Dry binder 0-2 2-5 2-5 3-8 3-8 (e.g., calcium silicate, kaolin) Fragrance, preservative QS QS QS QS QS Repeat sequence 2 2 2 2 2 protein polymer
TABLE-US-00052 WATER-IN-OIL FOUNDATION RAW MATERIAL (INCI Designation) Amount Cyclomethicone 12.0 Dimethicone 5.0 Cyclomethicone (and) Dimethicone copolyol 20.0 Laureth-7 0.5 Colorants (hydrophobically treated) 2.2 Titanium dioxide (and) methicone 8.5 Talc (and) methicone 3.3 Water 37.2 Sodium chloride 2.0 Propylene glycol 8.0 Fragrance, preservative QS Repeat sequence protein polymer 1
TABLE-US-00053 ANHYDROUS MAKEUP STICK RAW MATERIAL (INCI Designation) Amount Ozokerite wax 5.6 Polyethylene 5.3 Glyceryl dilaurate 5.5 Isostearyl neopentanoate 13.0 Octyldodecyl stearoyl stearate 12.0 Myristyl myristate 11.0 Ethylhexyl methoxycinnamate 7.5 PVP/Eicosene copolymer 0.5 Tocopherol acetate 0.1 Dimethicone (and) Trimethylsiloxysilicate 8.0 Cyclopentasiloxane 9.0 Mica 10.0 Talc 1.7 Titanium dioxide (and) Isopropyl titanium triisostearate 8.86 Iron oxides (and) Isopropyl titanium triisostearate 1.94 Fragrance, preservative QS Repeat sequence protein polymer 1.0
TABLE-US-00054 WATER-IN-SILICONE FOUNDATION RAW MATERIAL (INCI Designation) Amount Cetyl dimethicone copolyol 0.45 Polyglycerol-4 isostearate (and) Cetyl dimethicone 1.75 copolyol (and) Hexyl laurate Polyalkylene polysiloxane copolymer 0.9 Cetyl dimethicone 0.9 Beeswax 0.7 Castor wax (and) hydrogenated castor oil 0.35 Octyl palmitate 7.0 Cyclomethicone 7.95 Phenyl trimethicone 2.2 Titanium dioxide (and) Caprylyl silane 7.5 Iron oxides (and) Caprylyl silane 1.1 Talc (and) Caprylyl silane 3.8 Cyclomethicone 7.95 Dimethicone 1.3 Water 49.55 Sodium chloride 0.5 Propylene glycol 5.3 Fragrance, preservative QS Repeat sequence protein polymer 0.5
TABLE-US-00055 OIL-IN-WATER FOUNDATION RAW MATERIAL (INCI Designation) Amount Water 59.85 Polyvinylpyrrolidone 5.0 Magnesium aluminum silicate 2.0 Xanthan gum 0.4 Trisodium EDTA 0.05 Glyceryl polymethacrylate (and) Propylene glycol 1.0 (and) PVM/MA copolymer Polysorbate 20 1.0 Kaolin 0.8 Butylene glycol 4.0 Titanium dioxide 6.05 Iron oxides 1.15 Dimethicone 6.0 Ethylhexyl palmitate 2.0 PEG/PPG-25/25 Dimethicone 1.0 Tocopherol acetate 0.1 Retinyl palmitate 0.1 Silica 3.0 Cyclopentasiloxane 5.0 Fragrance, preservative QS Repeat sequence protein polymer 1.0
TABLE-US-00056 SUNSCREEN FORMULAS RAW MATERIAL Amount (INCI Designation) SPF ~25 SPF ~15 Water 52.65 71.10 PVM/MA decadiene crosspolymer 0.5 0.5 Butylene glycol 3.0 3.0 Disodium EDTA 0.1 0.1 PEG-20 stearate 1.5 1.5 Glyceryl stearate (and) Laureth-23 2.0 2.0 Isostearyl neopentanoate 1.0 1.0 Ethylhexyl palmitate 2.0 2.0 Glyceryl dilaurate 0.5 0.5 Octinoxate 7.5 7.5 Oxybenzone 2.0 2.0 Ethylhexyl salicylate 3.0 3.0 Sodium hydroxide (10%) 1.3 1.3 Glyceryl polymethacrylate (and) 3.0 3.0 Propylene glycol Glyceryl polymethacrylate (and) 0.5 0.5 Propylene glycol (and) PVM/MA copolymer Styrene/Acrylates copolymer (27% solids) 18.45 -- Fragrance, preservative QS QS Repeat sequence protein polymer 0.5 0.5
TABLE-US-00057 VERY WATER-RESISTANT SUNSCREEN FORMULAS RAW MATERIAL Amount (INCI Designation) SPF ~12 SPF ~22 Water 65.16 46.53 Acrylates copolymer 3.0 3.0 Disodium EDTA 0.1 0.1 Butylene glycol 2.0 2.0 Gylceryl polymethacrylate (and) 1.0 1.0 Propylene glycol (and) PVM/MA copolymer Butylated PVP 0.05 0.05 Glyceryl stearate (and) Behenyl alcohol 4.5 4.5 (and) Palmitic acid (and) Stearic acid (and) Lecithin (and) Lauryl alcohol Tricontanyl PVP 1.0 1.0 Octyl palmitate 2.0 2.0 Octinoxate 7.5 7.5 Oxybenzone 2.0 2.0 Ethylhexyl salicylate 3.0 3.0 Tridecyl neopentanoate 3.0 3.0 Glyceryl dilaurate 0.5 0.5 Sodium hydroxide (10%) 1.89 1.89 Cyclopentasiloxane 2.0 2.0 Butylene glycol 1.0 1.0 Styrene/Acrylates copolymer (27% solids) 18.45 -- Fragrance, preservative QS QS Repeat sequence protein polymer 0.5 0.5
TABLE-US-00058 WATER-IN-SILICONE SUNSCREEN RAW MATERIAL (INCI Designation) Amount Cetyl PEG/PPG-15/15 butyl ether dimethicone 2.0 Mineral oil 3.0 Ethylhexyl palmitate 1.0 Ethylhexyl salicylate 5.0 Hydrogenated castor oil 0.5 Beeswax 0.5 Octinoxate 7.5 Polyethylene 1.0 PEG-30 dipolyhydroxystearate 2.0 Cyclopentasiloxane 5.0 Dimethicone 5.0 Sodium chloride 0.6 Acrylates/C12-22 alkylmethacrylate copolymer 0.5 Water 66.4 Fragrance, preservative QS Repeat sequence protein polymer 0.5
Example 14
Foundation Formula
[0234] In this Example, a water in silicone foundation containing SELP47K is provided. This formulation contains the following ingredients:
TABLE-US-00059 Ingredient INCI Name % Water Phase Dow Corning 9011 Cyclopentasiloxane, 15.00 Elastomer Blend PEG-12 Dimethicone Copolymer Dow Corning 245 Fluid Cyclopentasiloxane 5.00 Silcare 31 M50 SV Caprylyl Trimethicone 6.35 Propylparaben 0.05 AS 5811 Titanium Dioxide, 7.50 Triethoxycaprylylsilane AS 5131 Iron Oxides, 0.70 Triethoxycaprylylsilane AS 5146 Iron Oxides, 0.05 Triethoxycaprylylsilane AS 5126 Iron Oxides, 0.35 Triethoxycaprylylsilane AS 50230 Talc, 3.50 Triethoxycaprylylsilane Oil Phase Deionized Water 53.30 SELP47K 1.80 Butylene Glycol 6.00 Methylparaben 0.20 Benzoic Acid 0.20 100.00
[0235] The pigments (AS 5811, 5131, 5146, 5126, and 50230; Color Techniques) and propylparaben were dispersed in Silcare 31 M50 SV (Clariant), stirring until wet. The mixture was then passed over a three roll mill at tight setting until particle size is <10 ฮผm. Then, DC 9011 Elastomer Blend (Dow Corning) and DC 245 Fluid were combined in finishing vessel, stirring until homogenous. The color grind was added with slow homogenizer agitation. The water was weighed into a separate vessel and SELP was gradually added with propeller agitation. stirring until dissolved. Methylparaben and benzoic acid were added to butylene glycol. The mixture was warmed slightly, and stirred until dissolved. The mixture was cooled to 30ยฐ C. and added to the SELP47K (GC2596-26-A) solution. The water phase was added slowly to the oil phase with rapid agitation. When addition was complete, the preparation was homogenized for five minutes. This preparation is useful as a makeup foundation for application to skin.
Example 15
Mascara Formulation
[0236] In this Example, a mascara formulation is provided. The ingredients of both a control preparation and a mascara containing 2% SELP47K (GC2596-26-A) are as follows:
TABLE-US-00060 % 2% Control SELP Trade Name INCI Name 1 2 Phase # Water Phase 1 Deionized Water 42.96 42.96 2 Butylene Glycol 5.00 5.00 2 Methylparaben 0.30 0.30 3 33-5198 (Black) Iron Oxides (Sun) 10.00 10.00 4 Natrosol 250 MR Hydroxyethylcellulose 0.20 0.20 (Aqualon) 5 10% KOH Potassium Hydroxide 0.01 0.01 6 Arlacel 165 Glyceryl Stearate, PEG-100 3.00 3.00 Stearate (Uniqema) 7 10% Citric Acid 0.27 0.27 Phase # Wax Phase 8 Arlacel 165 1.00 1.00 8 Cerasynt SD Glyceryl Stearate (ISP) 3.50 3.50 8 Beeswax, White SP 424 Beeswax (S&P) 7.50 7.50 8 Carnauba #1 Copernica Cerifera 4.80 4.80 (Carnauba) Wax (S&P) 8 Propylparaben 0.10 0.10 9 Deionized Water 20.00 -- 9 10% SELP/Water -- 20.00 10 Deionized Water 1.00 1.00 10 Glydant DMDM Hydantoin (Lonza) 0.36 0.36 100.00 100.00
[0237] To produce the mascara formulation, the wax phase 8 was combined and heated to 85-90ยฐ C. with propeller mixing. The 10% SELP47K solution was prepared by adding SELP47K powder to water while propeller mixing. Phase 1 water was added to a tared stainless steel beaker (approximately 50 g excess was added to compensate for loss). Phase 2 methylparaben was added to butylene glycol and stirred while warming on top of a steam bath until dissolved, then added to the water with slow homomixer agitation. Then, the phase 4 black iron oxide was added, while maintaining agitation. Then, Natrosol was sprinkled in, while maintaining agitation. The 10% KOH was added, and heating was begun to 85ยฐ C., with the beaker covered as tightly as possible. When the Natrosol was dissolved, the 10% citric acid was added dropwise, maintaining temperature and agitation. Then, the Arlacel 165 was added slowly and mixed for at least 5 minutes to insure dissolution. At 85-90ยฐ C., the wax phase was slowly added to the water phase while homomixing. The temperature and agitation are maintained for 10 minutes. The batch was removed from the steam bath and allowed to cool while homomixing with occasional hand scraping of the beaker walls. At 55ยฐ C., the batch was weighed to check for water loss. Mixing was resumed and water was added back, if necessary. At 45ยฐ C., phases 9 and 10 were added. Cooling was continued using cold water to 30ยฐ C. At this point, continuous hand scraping of beaker walls was necessary.
[0238] In this preparation, the small amount of KOH (in Phase 5) is used to raise the pH to disperse the Natrosol which is coated with glyoxal to retard wetting, and prevent agglomeration. In phase 7, the citric acid is added slowly to adjust pH to -5.5, below the isoelectric point of the iron oxides. In phases 7 and 8, the Arlacel 165 is split between the oil and water phases, as the emulsification is easier to accomplish with surfactant in both phases. In phase 9, the deionized water was added in the control batch instead of SELP47K. The SELP47K solution was prepared while the emulsion was being processed, so it was absolutely fresh. This preparation provides a formulation suitable for use as a mascara.
Example 16
Bioactivity Determination of RSPPs Using Human Skin Fibroblast Cells In-Vitro
[0239] In this Example, experiments conducted to determine bioactivity of repeat sequence protein polymers (RSPP) such as SELP47K and others (i.e., GC2596-26-A-E) in procollagen synthesis, matrix metaloprotease-1 (MMP-1) activity, and elastin production as well as elastase activity are described. The RSPP test materials used in these experiments are those shown in Table 1.
[0240] As dermal fibroblasts are the main source of extracellular matrix proteins, including large structural proteins such as collagen and elastin, as well as the enzymes that metabolize these proteins (e.g., Matrix Metalloprotease-1 [MMP-1] and elastase) within the skin, these experiments were conducted to determine bioactivity of RSPP in influencing collagen synthesis, elastin production, and MMP-1 activity. An accepted cultured human fibroblast model was used in these experiments.
[0241] Initial screening was conducted to identify a test range suitable to determine the effect of a broad range of test material concentrations on cell viability. In particular, these initial screening experiments were conducted in order to ensure that the concentrations of test RSPPs used within the study had a no cytotoxic effect. Cultured fibroblasts were exposed to the test materials for 48 hours and then changes in cell viability were determined using an MTT assay (i.e., a colorimetric analysis of the metabolic activity of the cultured cells, described in greater detail below).
[0242] When cells are treated with MTT, it is reduced by the mitochondria, which results in the formation of insoluble purple formazin crystals that are trapped within the cell. The purple crystals are extracted from the cells with isopropanol and the amount of crystal formation quantified spectrophotometrically. The intensity of the purple color is directly proportional to the metabolic activity of the cultured cells, which normally is used as an indication of an increase in the number of viable cells, although it can also indicate an increase in the mitochondrial content of the existing cells (Alley et al., Cancer Res., 48:589-601 1988). A decrease in color intensity can be a sign of toxicity.
[0243] Once an experimentally useful range of RSPP test material concentrations was determined, their effect on procollagen synthesis, elastin production and MMP-1 activity were then determined. All three of these markers are released into the cell culture media by the fibroblasts. Thus, after 48 hours of exposure to the test RSPP materials the media were collected and assayed.
[0244] Collagen is synthesized as part of a much larger procollagen peptide. As the peptide is processed to form a mature collagen protein, the propeptide portion is cleaved off (type I C-peptide). Both the mature collagen protein and the type I C-peptide fragment are then released into the extracellular environment and accumulate in the culture media. Since there is a 1:1 stoichiometric ratio between the two parts of the procollagen peptide, assaying for type I C-peptide will reflect the amount of collagen synthesized. Type 1 C-peptide can be assayed via an ELISA based method.
[0245] Elastin is released by fibroblasts (soluble elastin) into the extracellular space where it is then cross-linked to other elastin proteins to form an extensive network of fibers and sheets (insoluble elastin). Soluble elastin can be readily measured from cell culture medium via a colorimetric assay.
[0246] MMP-1 is a zinc and calcium dependent endopeptidase that is produced and released from both dermal fibroblasts and keratinocytes, and functions to break down collagens located in the extracellular matrix. Active MMP-1 released into the culture media can be assayed using a fluorescence-based ELISA. Briefly, antibodies covalently linked to a solid support will bind any MMP-1 (both active and inactive MMP-1) present in spent culture media samples. To quantify the active MMP-1 a fluorogenic substrate linked to a quencher molecule is added and any active MMP-1 present will cleave the peptide linkage between the fluorophore and the quencher molecule. This cleavage will eliminate the quencher molecules ability to inhibit the fluorescent signal of the fluorophore allowing a fluorescent signal that will be proportional to the amount of active MMP-1 present. The MMP-1 standards used with the ELISA kit (R&D Systems) used in the experiments described herein are provided in an inactive form and need to be activated by exposing them to p-aminophenylmercuric before adding the fluorogenic substrate.
[0247] The RSPP test materials were also tested to determine their effect(s) on elastase activity. In these experiments, human dermal fibroblasts were used as a source of the elastase enzyme. This enzyme was partially purified from the fibroblasts by lysing the cells in an elastase buffer and retaining the soluble portion of the lysate. Portions of this fibroblast lysate were then incubated with test materials and a synthetic elastase substrate, Suc-(Ala3)-p-Nitroaniline (STANA). Elastase acts upon this substrate to release p-nitroaniline, which can be detected spectrophotometrically by measuring the absorbance at a wavelength of 405 nm. An inhibition of the elastase enzyme is noted by a decrease in the amount of released p-nitroaniline when compared to uninhibited enzyme.
[0248] A. Procollagen Synthesis, MMP-1 Activity and Elastin Production By RSPP Test Materials
Preparation of Fibroblasts
[0249] Fibroblasts (Cascade Biologics, HDFn Cell Line, lot#1C0914) were seeded into the individual wells of either a 96-well plate (for the initial pilot work to determine concentration effects on cell viability) in 100 ฮผl of Fibroblast Growth Media (FGM) or in 12-well plates (for the measurement of collagen, elastin and MMP-1) in 1.0 ml of FGM and incubated overnight at 37ยฑ2ยฐ C. and 5ยฑ1% CO2. On the following day, the medium was removed via aspiration to eliminate any non-adherent cells and replaced with fresh FGM. The cells were grown until confluent, with a media change every 48 to 72 hours. Upon reaching confluency, the cells were treated for 24 hours with Dulbecco's Modified Essential Media (DMEM) supplemented with 1.5% FBS to wash out any effects from the growth factors included in the normal FGM. After this 24-hour wash out period, the cells were treated with the test materials at the specified concentrations dissolved in DMEM with 1.5% FBS. Untreated cells (negative controls) received DMEM with 1.5% FBS. The cells were incubated for 48 hours and at the end of the incubation period cell culture medium was either discarded (pilot work) or collected and stored frozen at -75ยฐ C. The test materials were tested in triplicate.
MTT Assay
[0250] After the 2-day incubation, the cell culture medium was removed and the fibroblasts were washed twice with PBS to remove any remaining test material. After the final wash, either 100 ฮผl (96-well plates) or 300 ฮผl (12-well plates) of DMEM supplemented with 0.5 mg/ml MTT were added to each well and the cells were incubated for 1 hour at 37ยฑ2ยฐ C. and 5ยฑ1% CO2. After the incubation, the DMEM/MTT solution was removed and the cells were washed again once with PBS and then either 100 ฮผl (96-well plates) or 1 ml (12-well plates) of isopropyl alcohol were added to each well to extract the purple formazin crystals. For the 12-well plates, two hundred microliters of the isopropyl extracts was transferred to a 96-well plate. The 96-well plates were read at 540 nm using isopropyl alcohol as a blank. The mean MTT absorbance value for the negative control cells was calculated and used to represent 100% value for cell viability. The individual MTT values from the cells undergoing the various treatments were then divided by the mean value for the negative control cells and expressed as a percent, to determine the change in cell viability caused by each treatment.
Procollagen Assay
[0251] Takara procollagen type IC-peptide ELISA kits were used in these experiments. A series of type I C-peptide standards was prepared ranging from 40 ng/ml to 640 ng/ml. An ELISA microplate was then prepared by removing any unneeded strips from the plate frame. To each well in the plate 100 ฮผl of peroxidase-labeled anti procollagen type I-C peptide was added, followed by 20 ฮผA of either sample (tissue culture media) or standard. The microplate was covered and allowed to incubate for 3ยฑ0.25 hours at 37ยฐ C. After the incubation period, each well was aspirated and washed three times with 400 ฮผl of wash buffer. After the last wash was removed, 100 ฮผl of peroxidase substrate solution (hydrogen peroxide containing tetramethylbenzidine as a chromagen) were added to each well and the plate was incubated for 15ยฑ5 minutes at room temperature. After the incubation period, 100 ฮผA of stop solution (1 N sulfuric acid) was added to each well and the plate was read using a microplate reader at 450 nm. To quantify the amount of procollagen present, a standard curve was generated using known concentrations of procollagen type 1-C peptide. A linear regression was then performed to establish the line that best fit these data points. Mean absorbance values for the test materials and untreated samples was then used to estimate the amount of procollagen type 1-C peptide present in each sample.
MMP-1 Assay
[0252] R&D Products MMP-1 Fluorokine E fluorometric ELISA kits were used in these experiments. A series of MMP-1 standards was prepared ranging from 0.39 ng/ml to 25 ng/ml and an ELISA microplate was prepared by removing any unneeded strips from the plate frame. In each well, 200 ฮผA of Assay Diluent RD1-64 and 50 ฮผA of each sample were added. For the standards, 100 ฮผl of Assay Diluent RD1-64 and 150 ฮผl of each respective standard were added. The plate was incubated for 3ยฑ0.25 hours at room temperature on a rocking platform. At the end of the incubation period, each well was aspirated and then washed four times with 400 ฮผl of wash buffer. Since the MMP-1 enzyme standards included with the assay kit are provided in an inactive form, after the last wash was removed, 200 ฮผl of p-aminophenylmercuric acetate in Reagent Dilution solution was added to the standards to activate MMP-1, while Reagent Dilution solution only was added to the samples and the plate was incubated for 2ยฑ0.25 hours at 37ยฑ2ยฐ C. After the incubation period, the plate was washed four times as described above. After the last wash, 200 ฮผl of substrate solution were added to each well and the plate was incubated for 17-20 hours at 37ยฑ2ยฐ C. (protected from light). At the end of the incubation period, the fluorescence intensity was measured (excitation 320 nm, emission 405 nm) using a fluorometer using a 20 mS integration time. To quantify the amount of MMP-1 activity, a standard curve was generated using known activity levels of MMP-1. A linear regression was performed to establish the line that best fit these data points. Mean absorbance values for the test materials and untreated samples were then used to estimate the amount of MMP-1 activity present in each sample.
Elastin Assay
[0253] In these experiments, the Biocolor Fastin Elastin Assay kits were used. A 50 ฮผl aliquot of sample material (cell culture medium for soluble elastin) was mixed with 1.0 ml of cold Fastin Precipitating Reagent in a 1.5-ml microcentrifuge tube. In addition to the spent media samples from the fibroblasts, 50 ฮผl aliquots of the test materials diluted into fresh culture media at the concentrations used in the study were also assayed for elastin content. The sample/Fastin Precipitating Reagent mixture was then incubated overnight in an ice water bath to precipitate the elastin. On the following day, the cold microcentrifuge tubes were spun at 10,000รg for 10 minutes to pack the precipitated elastin and the supernatant was removed via careful aspiration.
[0254] One (1) ml of Fastin Dye Reagent, along with 100 ฮผl of 90% saturated ammonium sulphate was added to each tube. The microcentrifuge tubes were mixed with a vortex mixer and then allowed to incubate for at least 60 minutes at room temperature, with gentle mechanical agitation. The Fastin Dye Reagent interacts with unique amino acid sequences in the elastin protein. This elastin-dye complex precipitates in the presence of ammonium sulphate. After the incubation was over, the microcentrifuge tubes were spun at 10,000รg for 10 minutes to separate the precipitated elastin-dye complex from the unbound dye. The supernatant was carefully removed and the tubes were inverted to allow any residual supernatant to drain out.
[0255] To release the elastin-bound dye, 1.0 ml of Fastin Destain reagent was added to each microcentrifuge tube. The tubes were capped as quickly as possible, as this reagent contains ammonia and methanol. The tubes were then vortexed. The absorbance of this solution was measured by a plate reader at 490 nm (200 ฮผl volume read).
[0256] An elastin standard curve was generated using 12.5, 25, and 50 ฮผg aliquots of an Elastin Standard into microcentrifuge tubes (in duplicate). These samples were treated as described above (Elastin Assay) and the absorbance values were used to generate a standard curve of known concentrations. To derive the standard curve for the elastin assay, the absorbance was plotted against the elastin concentration in ฮผg/ml for the standards. A linear regression was performed to establish the line that best fit these data points. Mean absorbance values for the test materials and untreated samples were then used to estimate the amount of elastin present in each sample.
[0257] B. Elastase Activity Experiments
Fibroblast Cell Culture Procedure
[0258] Fibroblasts (Cascade Biologics, HDFn Cell Line, lot#1C0914) were seeded in a 75-cm2 flask and grown in FGM and cultured at 37ยฑ2ยฐ C. and 5ยฑ1% CO2. Cells were expanded and passed until a sufficient number of cells had been grown (not exceeding 10 passages). When a sufficient number of cells had been grown, the fibroblast lysate containing the elastase enzyme was prepared as follows:
Fibroblast Lysate Procedure
[0259] The fibroblasts were washed twice with 15 ml of phosphate buffered saline with each wash removed via aspiration. Ice cold PBS (7.5 ml) was then added to the flask and the cells were detached with a cell scraper. The detached cells/PBS was transferred to a 15 ml centrifuge tube on ice. The flask was rinsed again with 7.5 ml of PBS, which was also transferred to the 15 ml tube. The tube was then centrifuged at 1,200 RPM for 5 minutes and the supernatant was removed via aspiration. The pellet of cells was resuspended in 2 ml of ice-cold 2ร elastase buffer and sonicated for 10 seconds (maximum setting) to lyse the cells. The sonication was repeated until the contents of the tube were clear while the tube was maintained on ice. The tube was then centrifuged at 2,200 RPM for 10 minutes at 4ยฐ C. and the supernatant was transferred to a new centrifuge tube (this portion of the fibroblast lysate contained the elastase enzyme) and kept on ice. The protein concentration of the fibroblast lysate was determined using the Pierce BCA Protein Assay kit (see below for details) and the fibroblast lysate was stored at -75ยฐ C. until used (for up to 1 year).
Bicinchoninic Acid (BCA) Protein Assay
[0260] Fifty volumes of Reagent A (BCA) were combined with 1 volume of Reagent B (4% (w/v) CuSO4-5H2O) in a 15-ml centrifuge tube. For the assay, proteins reduce Cu(II) to Cu(I) in a concentration dependent manner. BCA then reacts with the Cu(I) to form a purple colored complex with a maximum absorbance at 562 nm. Two hundred microliters of this combined reagent were dispensed into each well of a 96-well plate. Next, 10 ฮผl of each of the standards were added into their respective wells (standards were made using 2 mg/ml bovine serum albumin dissolved in elastase buffer, and then a series of 50% dilutions was made down to 0.0078 mg/ml). Ten ฮผl of elastase buffer was added to two wells to serve as blanks. The standards and blank were prepared in duplicate, while lysate samples were prepared in triplicate. The plate was covered, incubated it at 37ยฑ2ยฐ C. for 30ยฑ5 minutes and then read using a microplate reader at 540 nm.
Determination of Elastase Activity in Lysate-Range Finding
[0261] Upon determining the protein concentration of the lysate, a small sample was obtained and the elastase activity of the lysate was determined at 1ร, 0.5ร and 0.25ร concentrations. The activity was determined for each of the different concentrations in exactly the same way that the uninhibited sample was treated in the elastase assay (see below). First, 100 ฮผl of lysate was loaded into three wells, while 100 ฮผl of 2ร Elastase buffer was added to three additional wells to serve as blanks. Then, 100 ฮผl of diluted DMSO (1 part DMSO, 49 parts deionized water) was added to all six wells used and the 96-well plate was incubated for 15 minutes at 37ยฑ2ยฐ C. Then, 4 ฮผl of STANA was added to each well used, and the 96-well plate was returned to the incubator for 1.25 hours. After 1.25 hours, the well plate was removed and read in a 96-well plate reader at 405 nm. The mean absorbances were plotted versus concentration. These values were used to estimate a concentration that will produce a spectrophotometric measurement of 0.4 to 0.5 using a linear regression analysis of the best-fit line through all three (1ร, 0.5ร and 0.25ร) data points. The remaining fibroblast lysate was diluted to the appropriate concentration to elicit the desired level of activity.
Test Material Preparation
[0262] For this assay, test materials were prepared in deionized water at 2ร their final desired concentration. Since each material was tested at final concentrations of 1%, 0.5% and 0.1%, they were prepared at stock concentrations of 2%, 1% and 0.2%. 10 mM and 3 ฮผM phosphoramidon served as the positive control, while a 50 fold dilution of DMSO in deionized water served as a negative (uninhibited) control.
[0263] As these testing materials were hypothesized to potential inhibit the breakdown of STANA by the elastase enzyme via a competitive mechanism, the STANA substrate was mixed with the test materials so that they were added to the assay simultaneously. The STANA substrate was mixed with the stock test material solutions at a ratio of 4 ฮผl of STANA for every 100 ฮผl of stock test material solution.
Elastase Enzyme Assay, Modified Procedure
[0264] Six wells in a 96-well plate were reserved for each concentration of test material. In three of the wells 100 ฮผl of fibroblast lysate (lysate lot 082103) in 2ร elastase buffer were added, while in the three remaining wells, 100 ฮผl of 2ร elastase buffer alone were added. After the wells were prepared with lysate or buffer, 100 ฮผl of test material solution, phosphoramidon control, or diluted DMSO was added to the 6 wells reserved for each respective test. The plate was then covered and incubated at 37ยฑ2ยฐ C. The change in absorbance at 405 nm was measured at 0 minutes, 30 minutes, 60 minutes, 90 minutes and 120 minutes using a microplate reader.
[0265] For the elastase assay, the mean absorbance values for the three wells containing the various test materials and the fibroblast lysate were used as an index of elastase activity. The remaining three wells, containing the test materials and the elastase buffer were used to determine the amount of background absorbance due to either the test material itself or the non-specific breakdown of STANA. To correct the absorbance value associated with elastase activity, the background absorbance value was subtracted. To determine the percent of elastase inhibition induced by the test material, the following equation was used:
(1-(elastase activity with test material/elastase activity in untreated controls))ร100
[0266] C. Results
[0267] The results from the initial pilot cell viability dose response experiment are presented in Table 17.
TABLE-US-00061 TABLE 17 Initial Pilot Cell Viability Dose Response Results Test Material Concen- GC 2596- GC 2596- GC 2596- GC 2596- GC 2596- tration 26-A 26-B 26-C 26-D 26-E 1% 138 ยฑ 7 79 ยฑ 17 180 ยฑ 6 n/a 6 ยฑ 1 0.5% 171 ยฑ 4 120 ยฑ 5 180 ยฑ 12 n/a 17 ยฑ 4 0.14% 101 ยฑ 4 92 ยฑ 4 113 ยฑ 8 154 ยฑ 24 61 ยฑ 7 0.07% 96 ยฑ 6 101 ยฑ 3 107 ยฑ 10 145 ยฑ 11 75 ยฑ 9 0.014% 99 ยฑ 10 98 ยฑ 5 106 ยฑ 9 145 ยฑ 12 124 ยฑ 7 0.007% 101 ยฑ 5 92 ยฑ 10 97 ยฑ 8 138 ยฑ 9 113 ยฑ 6 0.0014% 99 ยฑ 1 99 ยฑ 11 107 ยฑ 10 130 ยฑ 15 117 ยฑ 10
[0268] The values are presented as a percent of untreated cells, which are used to represent 100% cell viability. A value above 100% indicates that the material either stimulated cell proliferation or an increase in mitochondrial activity. Based upon the results from this initial pilot work, suitable concentrations of the test materials were chosen for the remaining experiments.
[0269] The results from the MTT Assay are presented in Table 18. As in Table 17, the values are again presented as a percent of untreated cells, which are used to represent 100% cell viability. The results for the collagen synthesis assay are presented in Table 19, while the results for the MMP-1 activity assay are presented in Table 20. Finally, the results for elastin metabolism are presented in Table 21 (elastin production assay) and Table 22 (elastase inhibition assay). In these Tables, "A" represents GC2596-26-A, while "B" represents GC2596-26-B, "C" represents GC2596-26-C, "D" represents GC2596-26-D, and "E" represents GC2596-26-E.
TABLE-US-00062 TABLE 18 MTT Assay Results Treatment Percent Viability 1% A 120 ยฑ 4 0.5% A 122 ยฑ 3 0.1% A 98 ยฑ 3 1% B 77 ยฑ 4 0.5% B 95 ยฑ 2 0.1% B 102 ยฑ 4 1% C 127 ยฑ 3 0.5% C 123 ยฑ 4 0.1% C 102 ยฑ 4 0.014% D 106 ยฑ 5 0.007% D 103 ยฑ 2 0.0014% D 100 ยฑ 3 0.01% E 93 ยฑ 2 0.005% E 100 ยฑ 2 0.001% E 101 ยฑ 3
TABLE-US-00063 TABLE 19 Procollagen Assay Results Treatment Type-1 C Peptide (ng/ml) Untreated 873 ยฑ 111 1% A 687 ยฑ 49 0.5% A 715 ยฑ 72 0.1% A 932 ยฑ 116 1% B 990 ยฑ 40 0.5% B 1366 ยฑ 127 0.1% B 1370 ยฑ 169 1% C 714 ยฑ 120 0.5% C 900 ยฑ 143 0.1% C 817 ยฑ 55 0.014% D 829 ยฑ 171 0.007% D 908 ยฑ 94 0.0014% D 894 ยฑ 156 0.01% E 956 ยฑ 110 0.005% E 1162 ยฑ 194 0.001% E 838 ยฑ 75
TABLE-US-00064 TABLE 20 MMP-1 Assay Results Treatment MMP-1 Activity (ng/ml) Untreated 0.11 ยฑ 0.05 1% A 0.12 ยฑ 0.05 0.5% A 0.09 ยฑ 0.04 0.1% A 0.10 ยฑ 0.02 1% B 0.12 ยฑ 0.04 0.5% B 0.10 ยฑ 0.02 0.1% B 0.09 ยฑ 0.03 1% C 0.12 ยฑ 0.02 0.5% C 0.11 ยฑ 0.02 0.1% C 0.10 ยฑ 0.03 0.014% D -0.03 ยฑ 0.01 0.007% D -0.02 ยฑ 0.02 0.0014% D -0.01 ยฑ 0.01 0.01% E 0.00 ยฑ 0.02 0.005% E -0.01 ยฑ 0.02 0.001% E 0.04 ยฑ 0.05
TABLE-US-00065 TABLE 21 Elastin Assay Results Treatment Elastin (ฮผg/ml media) Untreated 69 ยฑ 2 1% A 360 ยฑ 67 0.5% A 102 ยฑ 10 0.1% A 61 ยฑ 16 1% B 43 ยฑ 12 0.5% B 47 ยฑ 9 0.1% B 67 ยฑ 7 1% C 63 ยฑ 15 0.5% C 70 ยฑ 10 0.1% C 68 ยฑ 7 0.014% D 68 ยฑ 10 0.007% D 69 ยฑ 4 0.0014% D 72 ยฑ 12 0.01% E 50 ยฑ 18 0.005% E 59 ยฑ 5 0.001% E 63 ยฑ 10 1% A media only -1 1% B media only -1 1% C media only -1 0.014% D media only -1 0.01% E media only -1
TABLE-US-00066 TABLE 22 Elastase Inhibition Assay Results Treatment Percent Inhibition 1% A -16 0.5% A -11 0.1% A -19 1% B -22 0.5% B -20 0.1% B -16 1% C 26 0.5% C 20 0.1% C 19 1% E -5 0.5% E -20 0.1% E -13 100 uM Phosphoramidon 96 30 nM Phosphoramidon 43
[0270] D. Summary of Data and Results
[0271] The initial dose response work (See, Table 17) with this set of peptides revealed a broad range of effects. Peptides GC2596-26-A, GC2596-26-C and GC2596-26-D were observed to strongly stimulate increases in the number of viable cells in a dose dependent manner. In contrast, peptides GC2596-26-B and GC2596-26-E) were observed to reduce the number of viable cells, with peptide B only having this effect at the highest concentration tested. GC2596-26-E had adverse effects at concentrations equal to or greater than 0.07%.
[0272] In view of these initial results, peptides GC2596-26-A, GC2596-26-B and GC2596-26-C were tested at 1%, 0.5% and 0.1% concentrations, while peptide GC2596-26-D was tested at 0.014%, 0.007% and 0.0014%, and GC2596-26-E was tested at 0.01%, 0.005% and 0.001% for the cell culture based assays. After 48 hours of incubation with the materials, the culture media were collected for analysis and the cells were again subjected to an MTT assay. The results for the second cell viability assay (See, Table 18) were confirmed the earlier results shown in Table 17, at the concentrations tested.
[0273] In the next set of experiments, the effect of the RSPP test materials on collagen synthesis was assessed (See, Table 19). Of these, RSPP test material GC2596-26-B showed improvement in collagen synthesis. Collagen synthesis was observed to be elevated at the 0.5% and 0.1% concentrations. However, at the 1% concentration, the amount of collagen synthesis declined back down to a level similar to the untreated group. The reason for this decline in collagen synthesis at the 1% concentration may possibly be related to the negative effect on cell viability that this material is also observed to have at this concentration, as shown in Table 23, below. Thus, when a ratio of collagen synthesis to viability is taken into account, RSPP test material GC2596-26-B improves collagen synthesis at all of the concentrations tested (i.e., as compared to the control).
TABLE-US-00067 TABLE 23 Test Results for Test Material GC2596-26-B Treatment Viability Score Collagen Score 1% B 77 990 0.5% B 95 1366 0.1% B 102 1370
[0274] Since the collagen content of the skin is regulated not only by the rate of collagen synthesis, but also on the rate of collagen breakdown, the effect of the materials on MMP-1 (collagenase) activity was also determined (See, Table 20). RSPP test materials GC2596-26-D and GC2596-26-E resulted in an almost complete loss of detectable MMP-1 activity in the samples. Although it is not intended that the present invention be limited to any particular mechanism, possible explanations include down-regulation of the gene expression of MMP-1, thereby shutting off the production of this enzyme. Another hypothesis is that the peptide prevented activation of any MMP-1 produced. Normally, MMP-1 is synthesized and released in an inactive form that must be activated extracellularly by MMP-3. If these two RSPP test materials were inhibiting MMP-3, then this would also have the effect of preventing MMP-1 activation.
[0275] Aside from collagen, another major component of the skins extracellular matrix is elastin. Like collagen, the elastin content of the skin is determined both by its rate of synthesis and its rate of degradation by elastase. With respect to elastin synthesis (See, Table 21), RSPP test material GC2596-26-A was observed to stimulate an excellent increase. On the degradation side (See, Table 22), RSPP test material GC2596-26-C was observed to inhibit the elastase enzyme, with an inhibition level of about 20%.
[0276] With respect to the elastin production assay, it should be noted that there was some concern that the RSPP test materials themselves might interfere with the assay since they contain sequences similar to elastin. Table 21 shows the results of assaying culture media supplemented with the highest concentrations of each of the test materials and for all 5 RSPP test materials there was no elastin detected. Thus, there appeared to be no interference with the assay methods due to the RSPP tested materials.
Example 17
UVB Protective Effect of SELP47K-P4 RSPP
[0277] This Example describes experiments conducted to assess the ability of SELP47K-P4 RSPP (GC 2596-26-D) to exert a protective effect by promoting cell survival after UVB exposure.
[0278] In these experiments, the test material was tested at six concentrations with two duplicate sets of cells prepared. The first set of cells was exposed to approximately 50 mj/cm2 of UVB, which has been shown to reduce the number of viable cells by up to 50%, followed immediately by the application of either the test material, 20 ฮผM Trolox (an anti-oxidant, used as a positive control), or left untreated (untreated controls). The second set of cells did not receive a dose of UVB and was just exposed to either the test material, Trolox, or left untreated. Changes in cell viability were then determined 48 hours post material application via an MTT assay, as described above.
[0279] As discussed in greater detail herein, the results suggested that the RSPP test material GC2596-26-D was capable of both improving cell survival when it was applied after UVB exposure, and also able to induce cell proliferation in a dose dependent manner. Therefore, the aim of the follow-up study, was to determine if treatment with the test material before or during UVB exposure provided any additional survival benefit(s). Four different treatment regiments were employed using different combinations of pretreatment with the test material prior to UVB exposure and treatment with the material during UVB exposure. Cell survival was again assessed via an MTT assay 48 hours after the UVB insult. In addition, this second study also incorporated the use of 2',7'-dichloro-dihydrofluorescein diacetate (DCF), which is a relatively non-fluorescent dye that can be loaded into cells and becomes fluorescent upon exposure to the reactive oxygen species (ROS) generated during UVB exposure (Carini et al., Il Farmaco 55:526-534
[2000]; and Chan et al., J. Cell. Biochem., 90:327-338
[2003]). The diacetate form of the dye can freely traverse cell membranes, however once inside of the cell intracellular esterases will cleave off the acetate side groups trapping the DCF dye within the cell. The results confirmed the UVB protective effect of the RSPP test material 2596-26-D when applied post UVB exposure, and also indicated that its protective effect was improved when the material was also present both before and/or during UVB exposure.
Preparation of Fibroblasts
[0280] Fibroblasts were seeded into the individual wells of a 96 well plate in 100 ฮผl FGM, and incubated overnight at 37ยฑ2ยฐ C. and 5ยฑ1% CO2. On the following day, the media were removed via aspiration to eliminate any non-adherent cells and replaced with 100 ฮผl of fresh FGM. The cells were grown until confluent, with a media change every 48 to 72 hours.
UVB Exposure and Treatment Protocol
[0281] An UVLM-26 lamp was used as the source of UVB light. UV light intensity was measured using a UVX radiometer coupled to a UVB sensor probe (UV Products) to determine the time required to deliver an approximate dose of 50 mj/cm2. Prior to UVB exposure, the FGM was replaced with 100 ฮผl PBS. After exposure, the PBS was removed and replaced with FGM supplemented with either the test materials, 20 ฮผM Trolox, or left unsupplemented. A duplicate set of cells was also prepared and treated as described above, with the exception that they were not exposed to UVB light. This second set was used to assess any changes in cell viability induced by the test materials alone. The cells were allowed to incubate with the test materials for 48 hours, and then changes in cell viability were determined via an MTT assay. The non-UVB exposed cells treated with unsupplemented FGM were used to represent 100% cell viability.
[0282] For this initial study the RSPP test material GC2596-26-D was tested at the following six combinations: 0.14%, 0.07%, 0.014%, 0.007%, 0.0014% and 0.0007%.
UVB Exposure and Pretreatment Protocol
[0283] Two sets of duplicate cells were prepared using the four treatment protocols outlined in Table 24. One set of cells was used for an MTT assay while the other set of cells was used to measure the formation of ROS using the fluorescent DCF dye indicator.
TABLE-US-00068 TABLE 24 Treatment Protocols 24 hour Material present pretreatment with Material present for 48 hours after Treatment test material during UVB exposure UVB exposure 1 + - + 2 + + + 3 - - + 4 - + +
[0284] In Table 24, "+" indicates that the material was present, while "-" indicates that the material was absent.
[0285] For this follow up study, the material was tested at concentrations of 0.14% and 0.014%. During the pretreatment phase, the test material was diluted in FGM (for treatments 1 and 2). Prior to UVB exposure the FGM was replaced with 100 ฮผl PBS (supplemented with the test material for treatments 2 and 4) using the same UVB exposure protocol as described above. After exposure, the PBS was removed and replaced with FGM supplemented with either the test materials or left unsupplemented. The cells were allowed to incubate with the test materials for 48 hours to assess changes in cell viability via an MTT assay and to determine changes in ROS formation via the fluorescent DCF dye indicator. For the MTT assay, cells that were not exposed to UVB or treated with test materials were used to represent 100% cell viability.
[0286] At the end of the 48 hour incubation period, the cells were photographed to record any changes in cell morphology.
MTT Assay
[0287] After the 48-hour incubation, the cell culture medium was removed and the fibroblasts were washed with PBS to remove any remaining test material. After the final wash, 100 ฮผl of DMEM supplemented with 0.5 mg/ml MTT was added to each well, and the cells were incubated for 1 to 2 hours at approx. 37ยฑ2ยฐ C. and 5ยฑ1% CO2. After the incubation period, the DMEM/MTT solution was removed and the cells were washed again, once with PBS and then 50 ฮผl of isopropyl alcohol was added to the well to extract the purple formazin crystals. The 96-well plate was then read at 540 nm using isopropyl alcohol as a blank.
[0288] The mean MTT absorbance value for the negative control cells (non-UVB exposed and non test material treated cells) was calculated and used to represent 100% value for cell number. The individual MTT values from the cells undergoing the various treatments was then divided by the mean value for the negative control cells and expressed as a percent, to determine the change in cell viability caused by each treatment. MTT scores for each treatment are expressed as meansยฑthe standard deviation of the mean.
[0289] The results of the initial MTT Assay for are presented in Table 25, below. The values are expressed as mean viabilityยฑstandard deviation with an n=4 for each treatment.
TABLE-US-00069 TABLE 25 Initial MTT Assay Results -UVB Percent +UVB Percent Treatment Viability Viability Untreated 100 ยฑ 6 55 ยฑ 9 0.14% 154 ยฑ 24 104 ยฑ 11 0.07% 145 ยฑ 11 90 ยฑ 20 0.014% 145 ยฑ 12 83 ยฑ 8 0.007% 138 ยฑ 9 81 ยฑ 12 0.0014% 130 ยฑ 15 76 ยฑ 13 0.0007% 131 ยฑ 7 69 ยฑ 9 20 ฮผM Trolox 120 ยฑ 11 77 ยฑ 9
[0290] The results for the follow-up MTT Assay are presented in Table 26. The values are expressed as mean viabilityยฑstandard deviation with an n=4 for each treatment. The T followed by a number indicates which treatment group the data belong to (See, Table 24).
TABLE-US-00070 TABLE 26 Follow-Up MTT Assay Results Treatment Percent Viability No UVB 100 ยฑ 27 UVB 79 ยฑ 19 T1 0.14% 122 ยฑ 20 T1 0.014% 85 ยฑ 7 T2 0.14% 126 ยฑ 25 T2 0.014% 78 ยฑ 4 T3 0.14% 97 ยฑ 14 T3 0.014% 88 ยฑ 17 T4 0.14% 121 ยฑ 19 T4 0.014% 86 ยฑ 6
Fluorescent DCF Dye Assay
[0291] One hour prior to UVB exposure, the cell culture media were removed and the wells were washed with PBS to remove any residual RSPP test material in the set of cells used for the DCF assay. A 20 mM stock of DCF prepared in DMSO was diluted to 10 ฮผM using PBS and 100 ฮผl of this solution were added to each well. The plate was then returned to the incubator for 1 hour, to allow sufficient time for the cells to take up and trap the dye. After the 1-hour incubation, the cells were washed with PBS to remove any unincorporated dye and then exposed to UVB using the protocol described above. Forty-eight hours later, the 96-well plate was read using a fluorometer set for an excitation wavelength of 485 nm and an emission wavelength of 518 nm. The duplicate set of wells run for the MTT assay served as non-dye loaded blanks to account for any background fluorescence due to the cells or the test material.
[0292] The mean fluorescence intensity was determined for each of the treatment groups (measured in relative fluorescence units or "RFU"). Since the test material was shown to induce cell proliferation, the RFU values were expressed as a ratio to the 48-hour MTT values, to normalize as best as possible for any changes in cell number that occurred.
[0293] The results for the DCF Assay are presented in Table 27. Values are expressed as the ratio of mean RFU to mean percent viability for each individual treatment.
TABLE-US-00071 TABLE 27 DCF Assay Results Treatment RFU/Viability No UVB 0.031 UVB 0.057 T1 0.14% 0.038 T1 0.014% 0.038 T2 0.14% 0.032 T2 0.014% 0.035 T3 0.14% 0.039 T3 0.014% 0.053 T4 0.14% 0.026 T4 0.014% 0.052
[0294] The purpose of these studies was to determine if RSPP test material GC2596-26-D describe above could provide protection against UVB-induced damage in cultured dermal fibroblasts. In the initial study, two duplicate sets of cultured fibroblasts were exposed to six concentrations of the test material. The first set of cells was exposed to approximately 50 mj/cm2 of UVB, followed immediately by the application of either the test material, 20 ฮผM Trolox (an anti-oxidant, used as a positive control), or left untreated (untreated controls). The second set of cells did not receive a dose of UVB and was only exposed to either the test material, Trolox, or left untreated. Changes in cell viability were then determined 48 hours post material application via an MTT assay.
Table 25 shows the results for this first study. With respect to the untreated cells, the dose of UVB was observed to induce an approximate 45% decrease in the number of viable cells (Untreated -UVB vs. Untreated +UVB). However, in the presence of the test material, this decrease in the number of viable cells induced by +UVB appears to be inhibited in a dose-dependent manner. This effect was most prominent at the highest concentration of test material used (0.14%), where the level of cell viability (104%) is nearly identical to the untreated -UVB exposed cells and almost twice the level of the untreated +UVB exposed cells. As the concentration of the test material decreased, so did the apparent protective effect, although even at the lowest level of the material, 0.0007%, the number of viable cells is still slightly greater than the +UVB untreated controls (69% vs. 55%, respectively).
[0295] It was also observed that in the absence of UVB light, RSPP test material GC2596-26-D could also stimulate cell proliferation in a dose dependent manner. At the highest concentration tested (0.14%), the number of viable cells in the -UVB condition was observed to be 54% greater than the -UVB untreated cells. This stimulation effect was apparent over the entire range of material D tested, with the level of proliferation decreasing as the concentration of the material decreased.
[0296] Although it is not intended that the present invention be limited to any particular mechanism, it is hypothesized that the material protects the cells by limiting the amount of damage or facilitates the repair of the damage. Thus, the population of viable cells is better maintained than it is in the untreated condition. This hypothesis is supported by the fact that the increase in the number of viable cells tends to be greater between the +UVB untreated and the +UVB 0.14% test material conditions (50% viability vs. 100% viability, respectively, double the number of viable cells) than in the -UVB untreated and the -UVB test material conditions (100% viability vs. 150% viability, respectively, only a 50% increase). This suggests that in the presence of UVB there is a greater increase in the number of viable cells than can be accounted for by purely stimulating cell proliferation in the surviving cells alone, and supports the explanation that the RSPP test material GC2596-26-D does provide protection against UVB induced damage.
[0297] Since the results of the initial MTT study suggested that the RSPP test material GC2596-26-D was capable of improving cell survival when it was applied after UVB exposure, a follow up study was conducted to determine if treatment with the test material before or during UVB exposure provided any additional survival benefit. Four different treatment regiments were employed using different combinations of pretreatment with the test material prior to UVB exposure and treatment with the material during UVB exposure. Cell survival was again assessed via an MTT assay 48 hours after the UVB insult. In addition, this second study also incorporated the use of 2',7'-dichloro-dihydrofluorescein diacetate (DCF), which is a relatively non-fluorescent dye that can be loaded into cells and becomes fluorescent upon exposure to the reactive oxygen species (ROS) generated during UVB exposure (Carini et al., supra; and Chan et al., supra). The diacetate form of the dye can freely traverse cell membranes, however once inside of the cell intracellular esterases will cleave off the acetate side groups trapping the DCF dye within the cell.
[0298] The results from the follow up experiments confirmed the UVB protective effect of the test material when applied post UVB exposure, and also indicated that its protective effect was improved when the material was also present both before and/or during UVB exposure. In this follow up study, Treatment 3 was identical to the treatment regimen used in the initial experiments, with the test material being applied after the UVB exposure, and shows similar results with respect to the MTT assay (See, Table 26). At the 0.14% concentration, cell viability after UVB exposure was again found to be close to 100% after the UVB exposure (vs. 104% in the initial study), and at the 0.014% concentration, cell viability was 88% (vs. 83% in the initial study). At both concentrations the amount of cell survival was greater than the amount of cell survival in the untreated cells. In addition, the use of the DCF dye allowed for an estimate of the amount of oxidative damage occurring within the cell as a result of the UVB exposure (See, Table 27). For this marker, the greater the value of the ratio, the greater the extent of the ROS formation within the cell. With respect to Treatment 3, both concentrations of the test material were observed to reduce the amount of ROS formed in a concentration-dependent manner, when compared to the +UVB control. These results confirm observations from the earlier study and further support that the test material 2596-26-D can provide UVB protection.
[0299] Treatments 1 and 2 incorporated a 24-hour pretreatment phase with the test material prior to the UVB exposure, with treatment 2 also having the test material present during the UVB exposure phase as well. When the material was present at 0.14%, cell viabilities in both treatments were greater than both the +UVB and -UVB controls. This may be mainly due to the fact that since this material stimulates cell proliferation, the cells undergoing the 24-hour pretreatment were growing at a faster rate than the rest of the cells in the experiment. However, the data from the DCF assay indicates that this pretreatment was effective in reducing the amount of ROS formation. Interestingly, the DCF assay also indicates that the material effectively reduced ROS formation at the 0.014% concentration, although the data from the MTT assay only shows a small increase in cell viability with this concentration of material D. Both Treatment 1 and 2 produced similar results, with Treatment 2 resulting in slightly less ROS formation, suggesting that the 24 hour pretreatment alone may be sufficient in providing protection and that having the material present again during UVB exposure may only provide minimal additional benefits.
[0300] The final Treatment (i.e., Treatment 4), involved having the material present during the UVB exposure. At 0.14% concentration, cell viability and ROS formation is comparable to Treatments 1 and 2 and clearly demonstrates a UVB protective effect. Thus it appears that having the material around at the 0.14% concentration either for 24 hour before exposure, or just during exposure provides a similar level of protection.
[0301] All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in cell biology, cosmetic formulations, protein biology, and molecular biology, as well as related fields are intended to be within the scope of the present invention.
Sequence CWU
1
1
3716PRTArtificial Sequencesynthetic silk-like protein 1Ser Gly Ala Gly Ala
Gly1 5259PRTUnknownsilk fibroin protein 2Gly Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala1 5
10 15Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala
Gly Ala Gly Ser Gly Ala 20 25
30Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser
35 40 45Gly Ala Gly Ala Gly Ser Gly Ala
Ala Gly Tyr 50 5535PRTArtificial Sequencesynthetic
elastin-like protein 3Gly Val Gly Val Pro1
5410PRTArtificial Sequencesynthetic abductin-like protein 4Gly Gly Phe
Gly Gly Met Gly Gly Gly Xaa1 5
1055PRTArtificial Sequencesynthetic byssus-like protein 5Gly Pro Gly Gly
Gly1 566PRTArtificial Sequencesynthetic gluten-like protein
6Pro Gly Gln Gly Gln Gln1 579PRTArtificial
Sequencesynthetic gluten-like protein 7Gly Tyr Tyr Pro Thr Ser Pro Gln
Gln1 583PRTArtificial Sequencesynthetic gluten-like protein
8Gly Gln Gln1928PRTArtificial Sequencesynthetic titin-like protein 9Pro
Pro Ala Lys Val Pro Glu Val Pro Lys Lys Pro Val Pro Glu Glu1
5 10 15Lys Val Pro Val Pro Val Pro
Lys Lys Pro Glu Ala 20 251012PRTArtificial
Sequencesynthetic extensin-like protein 10Ser Pro Pro Pro Pro Ser Pro Lys
Tyr Val Tyr Lys1 5 10114PRTArtificial
Sequencesynthetic fibronectin-like protein 11Arg Gly Asp
Ser1125PRTUnknowngliadin 12Pro Gln Gln Pro Tyr1
5135PRTUnknownglue polypeptide 13Pro Thr Thr Thr Lys1
5148PRTUnknownice nucleating protein 14Ala Gly Tyr Gly Ser Thr Gly Thr1
5158PRTUnknownkeratin 15Tyr Gly Gly Ser Ser Gly Gly Gly1
5165PRTUnknownkeratin 16Phe Gly Gly Gly Ser1
5176PRTUnknownmucin 17Thr Thr Thr Pro Asp Val1
5187PRTUnknownRNA polymerase II 18Tyr Ser Pro Thr Ser Pro Ser1
519780PRTUnknownSELP 47K 19Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
Ser Gly Val Gly Val1 5 10
15Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro
20 25 30Gly Val Gly Pro Gly Val Gly
Pro Gly Val Gly Pro Gly Val Gly Pro 35 40
45Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
Ala 50 55 60Gly Ser Gly Ala Gly Ala
Gly Ser Gly Val Gly Val Pro Gly Val Gly65 70
75 80Val Pro Gly Val Gly Val Pro Gly Lys Gly Val
Pro Gly Val Gly Pro 85 90
95Gly Val Gly Pro Gly Val Gly Pro Gly Val Gly Pro Gly Ala Gly Ala
100 105 110Gly Ser Gly Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala 115 120
125Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro
Gly Val 130 135 140Gly Val Pro Gly Lys
Gly Val Pro Gly Val Gly Pro Gly Val Gly Pro145 150
155 160Gly Val Gly Pro Gly Val Gly Pro Gly Ala
Gly Ala Gly Ser Gly Ala 165 170
175Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser
180 185 190Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 195
200 205Lys Gly Val Pro Gly Val Gly Pro Gly Val Gly Pro
Gly Val Gly Pro 210 215 220Gly Val Gly
Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser225
230 235 240Gly Ala Gly Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly Val Gly Val 245
250 255Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
Lys Gly Val Pro 260 265 270Gly
Val Gly Pro Gly Val Gly Pro Gly Val Gly Pro Gly Val Gly Pro 275
280 285Gly Ala Gly Ala Gly Ser Gly Ala Gly
Ala Gly Ser Gly Ala Gly Ala 290 295
300Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly305
310 315 320Val Pro Gly Val
Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Pro 325
330 335Gly Val Gly Pro Gly Val Gly Pro Gly Val
Gly Pro Gly Ala Gly Ala 340 345
350Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala
355 360 365Gly Ala Gly Ser Gly Val Gly
Val Pro Gly Val Gly Val Pro Gly Val 370 375
380Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Pro Gly Val Gly
Pro385 390 395 400Gly Val
Gly Pro Gly Val Gly Pro Gly Ala Gly Ala Gly Ser Gly Ala
405 410 415Gly Ala Gly Ser Gly Ala Gly
Ala Gly Ser Gly Ala Gly Ala Gly Ser 420 425
430Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
Pro Gly 435 440 445Lys Gly Val Pro
Gly Val Gly Pro Gly Val Gly Pro Gly Val Gly Pro 450
455 460Gly Val Gly Pro Gly Ala Gly Ala Gly Ser Gly Ala
Gly Ala Gly Ser465 470 475
480Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val
485 490 495Pro Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro 500
505 510Gly Val Gly Pro Gly Val Gly Pro Gly Val Gly Pro
Gly Val Gly Pro 515 520 525Gly Ala
Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala 530
535 540Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
Val Pro Gly Val Gly545 550 555
560Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Pro
565 570 575Gly Val Gly Pro
Gly Val Gly Pro Gly Val Gly Pro Gly Ala Gly Ala 580
585 590Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
Ala Gly Ser Gly Ala 595 600 605Gly
Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 610
615 620Gly Val Pro Gly Lys Gly Val Pro Gly Val
Gly Pro Gly Val Gly Pro625 630 635
640Gly Val Gly Pro Gly Val Gly Pro Gly Ala Gly Ala Gly Ser Gly
Ala 645 650 655Gly Ala Gly
Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser 660
665 670Gly Val Gly Val Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly 675 680
685Lys Gly Val Pro Gly Val Gly Pro Gly Val Gly Pro Gly Val Gly Pro 690
695 700Gly Val Gly Pro Gly Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly Ser705 710
715 720Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser
Gly Val Gly Val 725 730
735Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro
740 745 750Gly Val Gly Pro Gly Val
Gly Pro Gly Val Gly Pro Gly Val Gly Pro 755 760
765Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser 770
775 780203PRTArtificial Sequencesynthetic
abductin-like protein 20Gly Xaa Xaa12139DNAArtificial Sequenceprimer
21gggagttggt gtacctggag aaggtgttcc gggggtagg
392239DNAArtificial Sequenceprimer 22cctacccccg gaacaccttc tccaggtaca
ccaactccc 392339DNAArtificial Sequenceprimer
23gggagttggg gtacctggac gaggtgttcc gggggtagg
392440DNAArtificial Sequenceprimer 24cctacccccc caaggtggag ctccaggtac
cccaactccc 4025884PRTUnknownSELP 47E-13 25Met
Asp Pro Val Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val1
5 10 15Thr Gln Leu Asn Arg Leu Ala
Ala His Pro Pro Phe Ala Ser Asp Pro 20 25
30Met Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
Val Pro 35 40 45Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 50 55
60Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Glu
Pro Gly Val65 70 75
80Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
85 90 95Ala Gly Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly Val Gly Val Pro 100
105 110Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly 115 120 125Glu Gly
Val Pro Gly Val Gly Val Pro Gly Val Gly Glu Pro Gly Val 130
135 140Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala
Gly Ala Gly Ser Gly145 150 155
160Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro
165 170 175Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180
185 190Glu Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Glu Pro Gly Val 195 200 205Gly
Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 210
215 220Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
Ser Gly Val Gly Val Pro225 230 235
240Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
Gly 245 250 255Glu Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly Glu Pro Gly Val 260
265 270Gly Val Pro Gly Ala Gly Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly 275 280
285Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro 290
295 300Gly Val Gly Val Pro Gly Val Gly
Val Pro Gly Val Gly Val Pro Gly305 310
315 320Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
Glu Pro Gly Val 325 330
335Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
340 345 350Ala Gly Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly Val Gly Val Pro 355 360
365Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
Pro Gly 370 375 380Glu Gly Val Pro Gly
Val Gly Val Pro Gly Val Gly Glu Pro Gly Val385 390
395 400Gly Val Pro Gly Ala Gly Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly 405 410
415Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro
420 425 430Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 435
440 445Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
Glu Pro Gly Val 450 455 460Gly Val Pro
Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly465
470 475 480Ala Gly Ala Gly Ser Gly Ala
Gly Ala Gly Ser Gly Val Gly Val Pro 485
490 495Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly 500 505 510Glu
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Glu Pro Gly Val 515
520 525Gly Val Pro Gly Ala Gly Ala Gly Ser
Gly Ala Gly Ala Gly Ser Gly 530 535
540Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro545
550 555 560Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 565
570 575Glu Gly Val Pro Gly Val Gly Val Pro Gly
Val Gly Glu Pro Gly Val 580 585
590Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
595 600 605Ala Gly Ala Gly Ser Gly Ala
Gly Ala Gly Ser Gly Val Gly Val Pro 610 615
620Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
Gly625 630 635 640Glu Gly
Val Pro Gly Val Gly Val Pro Gly Val Gly Glu Pro Gly Val
645 650 655Gly Val Pro Gly Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly Ser Gly 660 665
670Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
Val Pro 675 680 685Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 690
695 700Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
Glu Pro Gly Val705 710 715
720Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
725 730 735Ala Gly Ala Gly Ser
Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro 740
745 750Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly 755 760 765Glu Gly
Val Pro Gly Val Gly Val Pro Gly Val Gly Glu Pro Gly Val 770
775 780Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala
Gly Ala Gly Ser Gly785 790 795
800Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro
805 810 815Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 820
825 830Glu Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Glu Pro Gly Val 835 840 845Gly
Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 850
855 860Ala Gly Ala Met Asp Pro Gly Arg Tyr Gln
Asp Leu Arg Ser His His865 870 875
880His His His His26246PRTUnknownSELP 47R-3 26Met Pro Pro Val
Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val1 5
10 15Thr Gln Leu Asn Arg Leu Ala Ala His Pro
Pro Phe Ala Ser Asp Pro 20 25
30Met Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
35 40 45Val Pro Gly Val Gly Val Pro Gly
Val Gly Val Pro Gly Val Gly Val 50 55
60Pro Gly Arg Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro65
70 75 80Gly Val Gly Val Pro
Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 85
90 95Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala
Gly Ser Gly Val Gly 100 105
110Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
115 120 125Pro Gly Arg Gly Val Pro Gly
Val Gly Val Pro Gly Val Gly Val Pro 130 135
140Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala
Gly145 150 155 160Ser Gly
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
165 170 175Val Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly Val Gly Val 180 185
190Pro Gly Arg Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro 195 200 205Gly Val Gly Val
Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 210
215 220Ser Gly Ala Gly Ala Met Asp Pro Gly Arg Tyr Gln
Asp Leu Arg Ser225 230 235
240His His His His His His 24527244PRTUnknownSELP-47K-3
27Met Asp Pro Val Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val1
5 10 15Thr Gln Leu Asn Arg Leu
Ala Ala His Pro Pro Phe Ala Ser Asp Pro 20 25
30Met Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val
Gly Val Pro 35 40 45Gly Val Gly
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 50
55 60Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro Gly Val65 70 75
80Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
85 90 95Ala Gly Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly Val Gly Val Pro 100
105 110Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly 115 120 125Lys Gly
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 130
135 140Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala
Gly Ala Gly Ser Gly145 150 155
160Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro
165 170 175Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180
185 190Lys Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly Val 195 200 205Gly
Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 210
215 220Ala Gly Ala Met Asp Pro Gly Arg Tyr Gln
Asp Leu Arg Ser His His225 230 235
240His His His His28246PRTUnknownSELP 47E-3 28Met Asp Pro Val
Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val1 5
10 15Thr Gln Leu Asn Arg Leu Ala Ala His Pro
Pro Phe Ala Ser Asp Pro 20 25
30Met Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
35 40 45Val Pro Gly Val Gly Val Pro Gly
Val Gly Val Pro Gly Val Gly Val 50 55
60Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro65
70 75 80Gly Val Gly Val Pro
Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 85
90 95Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala
Gly Ser Gly Val Gly 100 105
110Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
115 120 125Pro Gly Glu Gly Val Pro Gly
Val Gly Val Pro Gly Val Gly Val Pro 130 135
140Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala
Gly145 150 155 160Ser Gly
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
165 170 175Val Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly Val Gly Val 180 185
190Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro 195 200 205Gly Val Gly Val
Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 210
215 220Ser Gly Ala Gly Ala Met Asp Pro Gly Arg Tyr Gln
Asp Leu Arg Ser225 230 235
240His His His His His His 245291063PRTArtificial
Sequencesynthetic collagen-like protein 29Met Asp Pro Val Val Leu Gln Arg
Arg Asp Trp Glu Asn Pro Gly Val1 5 10
15Thr Gln Leu Asn Arg Leu Ala Ala His Pro Pro Phe Ala Ser
Asp Pro 20 25 30Met Gly Ala
His Gly Pro Ala Gly Pro Lys Gly Ala His Gly Pro Ala 35
40 45Gly Pro Lys Gly Ala Gln Gly Pro Ala Gly Pro
Gly Gly Ala Gln Gly 50 55 60Pro Ala
Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala65
70 75 80Gln Gly Pro Ala Gly Pro Gly
Gly Ala Gln Gly Pro Ala Gly Pro Gly 85 90
95Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly
Pro Ala Gly 100 105 110Pro Gly
Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro 115
120 125Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala
Gly Pro Gly Gly Ala Gln 130 135 140Gly
Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly145
150 155 160Ala Gln Gly Pro Ala Gly
Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro 165
170 175Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala
Gln Gly Pro Ala 180 185 190Gly
Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly 195
200 205Pro Ala Gly Pro Gly Gly Ala Gln Gly
Pro Ala Gly Pro Gly Gly Ala 210 215
220Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly225
230 235 240Gly Ala Gln Gly
Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly 245
250 255Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro
Gly Gly Ala His Gly Pro 260 265
270Ala Gly Pro Lys Gly Ala His Gly Pro Ala Gly Pro Lys Gly Ala His
275 280 285Gly Pro Ala Gly Pro Lys Gly
Ala His Gly Pro Ala Gly Pro Lys Gly 290 295
300Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly
Pro305 310 315 320Gly Gly
Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala
325 330 335Gly Pro Gly Gly Ala Gln Gly
Pro Ala Gly Pro Gly Gly Ala Gln Gly 340 345
350Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly
Gly Ala 355 360 365Gln Gly Pro Ala
Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly 370
375 380Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln
Gly Pro Ala Gly385 390 395
400Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro
405 410 415Ala Gly Pro Gly Gly
Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln 420
425 430Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala
Gly Pro Gly Gly 435 440 445Ala Gln
Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro 450
455 460Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly
Ala Gln Gly Pro Ala465 470 475
480Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly
485 490 495Pro Ala Gly Pro
Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala 500
505 510Gln Gly Pro Ala Gly Pro Gly Gly Ala His Gly
Pro Ala Gly Pro Lys 515 520 525Gly
Ala His Gly Pro Ala Gly Pro Lys Gly Ala His Gly Pro Ala Gly 530
535 540Pro Lys Gly Ala His Gly Pro Ala Gly Pro
Lys Gly Ala Gln Gly Pro545 550 555
560Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala
Gln 565 570 575Gly Pro Ala
Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly 580
585 590Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala
Gln Gly Pro Ala Gly Pro 595 600
605Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala 610
615 620Gly Pro Gly Gly Ala Gln Gly Pro
Ala Gly Pro Gly Gly Ala Gln Gly625 630
635 640Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly
Pro Gly Gly Ala 645 650
655Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly
660 665 670Gly Ala Gln Gly Pro Ala
Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly 675 680
685Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln
Gly Pro 690 695 700Ala Gly Pro Gly Gly
Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln705 710
715 720Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly
Pro Ala Gly Pro Gly Gly 725 730
735Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro
740 745 750Gly Gly Ala Gln Gly
Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala 755
760 765Gly Pro Gly Gly Ala His Gly Pro Ala Gly Pro Lys
Gly Ala His Gly 770 775 780Pro Ala Gly
Pro Lys Gly Ala His Gly Pro Ala Gly Pro Lys Gly Ala785
790 795 800His Gly Pro Ala Gly Pro Lys
Gly Ala Gln Gly Pro Ala Gly Pro Gly 805
810 815Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln
Gly Pro Ala Gly 820 825 830Pro
Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro 835
840 845Ala Gly Pro Gly Gly Ala Gln Gly Pro
Ala Gly Pro Gly Gly Ala Gln 850 855
860Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly865
870 875 880Ala Gln Gly Pro
Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro 885
890 895Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly
Gly Ala Gln Gly Pro Ala 900 905
910Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly
915 920 925Pro Ala Gly Pro Gly Gly Ala
Gln Gly Pro Ala Gly Pro Gly Gly Ala 930 935
940Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro
Gly945 950 955 960Gly Ala
Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly
965 970 975Pro Gly Gly Ala Gln Gly Pro
Ala Gly Pro Gly Gly Ala Gln Gly Pro 980 985
990Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly
Ala Gln 995 1000 1005Gly Pro Ala
Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly 1010
1015 1020Ala His Gly Pro Ala Gly Pro Lys Gly Ala His Gly
Pro Ala Gly Pro1025 1030 1035
1040Lys Met Asp Pro Gly Arg Tyr Gln Leu Ser Ala Gly Arg Tyr His Tyr
1045 1050 1055Gln Leu Val Trp Cys
Gln Lys 1060301038PRTUnknownSELP 67K 30Met Asp Pro Val Val Leu
Gln Arg Arg Asp Trp Glu Asn Pro Gly Val1 5
10 15Thr Gln Leu Asn Arg Leu Ala Ala His Pro Pro Phe
Ala Ser Asp Pro 20 25 30Met
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 35
40 45Gly Val Gly Val Pro Gly Lys Gly Val
Pro Gly Val Gly Val Pro Gly 50 55
60Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly65
70 75 80Ala Gly Ala Gly Ser
Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 85
90 95Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala
Gly Ser Gly Val Gly 100 105
110Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
115 120 125Pro Gly Lys Gly Val Pro Gly
Val Gly Val Pro Gly Val Gly Val Pro 130 135
140Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala
Gly145 150 155 160Ser Gly
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
165 170 175Ala Gly Ser Gly Ala Gly Ala
Gly Ser Gly Val Gly Val Pro Gly Val 180 185
190Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
Lys Gly 195 200 205Val Pro Gly Val
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 210
215 220Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
Ser Gly Ala Gly225 230 235
240Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
245 250 255Ala Gly Ala Gly Ser
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 260
265 270Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly
Val Pro Gly Val 275 280 285Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly 290
295 300Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala
Gly Ala Gly Ser Gly305 310 315
320Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
325 330 335Ser Gly Val Gly
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 340
345 350Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly
Val Gly Val Pro Gly 355 360 365Val
Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly 370
375 380Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
Ser Gly Ala Gly Ala Gly385 390 395
400Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val
Gly 405 410 415Val Pro Gly
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 420
425 430Pro Gly Lys Gly Val Pro Gly Val Gly Val
Pro Gly Val Gly Val Pro 435 440
445Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 450
455 460Ser Gly Ala Gly Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly Ala Gly465 470
475 480Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly
Val Pro Gly Val 485 490
495Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly
500 505 510Val Pro Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly Val 515 520
525Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
Ala Gly 530 535 540Ala Gly Ser Gly Ala
Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly545 550
555 560Ala Gly Ala Gly Ser Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly 565 570
575Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val
580 585 590Gly Val Pro Gly Val
Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly 595
600 605Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
Ala Gly Ser Gly 610 615 620Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly625
630 635 640Ser Gly Val Gly Val Pro Gly
Val Gly Val Pro Gly Val Gly Val Pro 645
650 655Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val
Gly Val Pro Gly 660 665 670Val
Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly 675
680 685Ala Gly Ala Gly Ser Gly Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly 690 695
700Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly705
710 715 720Val Pro Gly Val
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 725
730 735Pro Gly Lys Gly Val Pro Gly Val Gly Val
Pro Gly Val Gly Val Pro 740 745
750Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
755 760 765Ser Gly Ala Gly Ala Gly Ser
Gly Ala Gly Ala Gly Ser Gly Ala Gly 770 775
780Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly
Val785 790 795 800Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly
805 810 815Val Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly Val Gly Val 820 825
830Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
Ala Gly 835 840 845Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 850
855 860Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly865 870 875
880Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val
885 890 895Gly Val Pro Gly Val
Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly 900
905 910Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
Ala Gly Ser Gly 915 920 925Ala Gly
Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 930
935 940Ser Gly Val Gly Val Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro945 950 955
960Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly
965 970 975Val Gly Val Pro
Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly 980
985 990Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser
Gly Ala Gly Ala Gly 995 1000
1005Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Met Asp Pro
1010 1015 1020Gly Arg Tyr Gln Asp Leu Arg
Ser His His His His His His1025 1030
103531965PRTArtificial SequenceSELP 58 31Met Asp Pro Val Val Leu Gln Arg
Arg Asp Trp Glu Asn Pro Gly Val1 5 10
15Thr Gln Leu Asn Arg Leu Ala Ala His Pro Pro Phe Ala Ser
Asp Pro 20 25 30Met Gly Val
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 35
40 45Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
Val Gly Val Pro Gly 50 55 60Val Gly
Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly65
70 75 80Ala Gly Ala Gly Ser Gly Ala
Gly Ala Gly Ser Gly Ala Gly Ala Gly 85 90
95Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly
Val Gly Val 100 105 110Pro Gly
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 115
120 125Gly Val Gly Val Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly 130 135 140Ala
Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly145
150 155 160Ser Gly Ala Gly Ala Gly
Ser Gly Ala Gly Ala Gly Ser Gly Val Gly 165
170 175Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
Gly Val Gly Val 180 185 190Pro
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 195
200 205Gly Val Gly Val Pro Gly Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly 210 215
220Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly225
230 235 240Ala Gly Ser Gly
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 245
250 255Val Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro Gly Val Gly Val 260 265
270Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly
275 280 285Ser Gly Ala Gly Ala Gly Ser
Gly Ala Gly Ala Gly Ser Gly Ala Gly 290 295
300Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly
Val305 310 315 320Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
325 330 335Val Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly Val Gly Val 340 345
350Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
Ala Gly 355 360 365Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 370
375 380Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro Gly Val385 390 395
400Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
405 410 415Val Pro Gly Val Gly
Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly 420
425 430Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
Ala Gly Ser Gly 435 440 445Ala Gly
Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 450
455 460Val Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly Val465 470 475
480Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly
485 490 495Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 500
505 510Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser
Gly Val Gly Val Pro 515 520 525Gly
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 530
535 540Val Gly Val Pro Gly Val Gly Val Pro Gly
Val Gly Val Pro Gly Val545 550 555
560Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser
Gly 565 570 575Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 580
585 590Ser Gly Val Gly Val Pro Gly Val Gly Val
Pro Gly Val Gly Val Pro 595 600
605Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 610
615 620Val Gly Val Pro Gly Val Gly Val
Pro Gly Ala Gly Ala Gly Ser Gly625 630
635 640Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
Ala Gly Ala Gly 645 650
655Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val
660 665 670Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly Val Gly Val Pro 675 680
685Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
Pro Gly 690 695 700Ala Gly Ala Gly Ser
Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly705 710
715 720Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
Ala Gly Ser Gly Val Gly 725 730
735Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
740 745 750Pro Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 755
760 765Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly
Ala Gly Ala Gly 770 775 780Ser Gly Ala
Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly785
790 795 800Ala Gly Ser Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly 805
810 815Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
Gly Val Gly Val 820 825 830Pro
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly 835
840 845Ser Gly Ala Gly Ala Gly Ser Gly Ala
Gly Ala Gly Ser Gly Ala Gly 850 855
860Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val865
870 875 880Gly Val Pro Gly
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 885
890 895Val Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro Gly Val Gly Val 900 905
910Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
915 920 925Ala Gly Ser Gly Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly Ser Met 930 935
940Asp Pro Gly Arg Tyr Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln
Leu945 950 955 960Val Trp
Cys Gln Lys 965321016PRTUnknownSELP 47K-P4 32Met Asp Pro
Val Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val1 5
10 15Thr Gln Leu Asn Arg Leu Ala Ala His
Pro Pro Phe Ala Ser Asp Pro 20 25
30Met Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Ala Leu Ser
35 40 45Tyr Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly Val Gly Val 50 55
60Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro65
70 75 80Gly Val Gly Val
Pro Gly Val Gly Val Pro Ala Leu Ser Tyr Pro Gly 85
90 95Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
Ser Gly Ala Gly Ala Gly 100 105
110Ser Gly Ala Gly Ala Gly Ser Ala Leu Ser Tyr Pro Gly Val Gly Val
115 120 125Pro Gly Val Gly Val Pro Gly
Val Gly Val Pro Gly Val Gly Val Pro 130 135
140Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
Gly145 150 155 160Val Gly
Val Pro Ala Leu Ser Tyr Pro Gly Ala Gly Ala Gly Ser Gly
165 170 175Ala Gly Ala Gly Ser Gly Ala
Gly Ala Gly Ser Gly Ala Gly Ala Gly 180 185
190Ser Ala Leu Ser Tyr Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro 195 200 205Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly 210
215 220Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro Ala Leu225 230 235
240Ser Tyr Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
245 250 255Ala Gly Ala Gly Ser
Gly Ala Gly Ala Gly Ser Ala Leu Ser Tyr Pro 260
265 270Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly 275 280 285Val Gly
Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val 290
295 300Gly Val Pro Gly Val Gly Val Pro Ala Leu Ser
Tyr Pro Gly Ala Gly305 310 315
320Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly
325 330 335Ala Gly Ala Gly
Ser Ala Leu Ser Tyr Pro Gly Val Gly Val Pro Gly 340
345 350Val Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly Lys 355 360 365Gly
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 370
375 380Val Pro Ala Leu Ser Tyr Pro Gly Ala Gly
Ala Gly Ser Gly Ala Gly385 390 395
400Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser
Ala 405 410 415Leu Ser Tyr
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 420
425 430Gly Val Pro Gly Val Gly Val Pro Gly Lys
Gly Val Pro Gly Val Gly 435 440
445Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Ala Leu Ser Tyr 450
455 460Pro Gly Ala Gly Ala Gly Ser Gly
Ala Gly Ala Gly Ser Gly Ala Gly465 470
475 480Ala Gly Ser Gly Ala Gly Ala Gly Ser Ala Leu Ser
Tyr Pro Gly Val 485 490
495Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
500 505 510Val Pro Gly Lys Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly Val 515 520
525Pro Gly Val Gly Val Pro Ala Leu Ser Tyr Pro Gly Ala Gly
Ala Gly 530 535 540Ser Gly Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly545 550
555 560Ala Gly Ser Ala Leu Ser Tyr Pro Gly Val
Gly Val Pro Gly Val Gly 565 570
575Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val
580 585 590Pro Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 595
600 605Ala Leu Ser Tyr Pro Gly Ala Gly Ala Gly Ser Gly
Ala Gly Ala Gly 610 615 620Ser Gly Ala
Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Ala Leu Ser625
630 635 640Tyr Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly Val Gly Val 645
650 655Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly
Val Gly Val Pro 660 665 670Gly
Val Gly Val Pro Gly Val Gly Val Pro Ala Leu Ser Tyr Pro Gly 675
680 685Ala Gly Ala Gly Ser Gly Ala Gly Ala
Gly Ser Gly Ala Gly Ala Gly 690 695
700Ser Gly Ala Gly Ala Gly Ser Ala Leu Ser Tyr Pro Gly Val Gly Val705
710 715 720Pro Gly Val Gly
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 725
730 735Gly Lys Gly Val Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly 740 745
750Val Gly Val Pro Ala Leu Ser Tyr Pro Gly Ala Gly Ala Gly Ser Gly
755 760 765Ala Gly Ala Gly Ser Gly Ala
Gly Ala Gly Ser Gly Ala Gly Ala Gly 770 775
780Ser Ala Leu Ser Tyr Pro Gly Val Gly Val Pro Gly Val Gly Val
Pro785 790 795 800Gly Val
Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly
805 810 815Val Gly Val Pro Gly Val Gly
Val Pro Gly Val Gly Val Pro Ala Leu 820 825
830Ser Tyr Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly
Ser Gly 835 840 845Ala Gly Ala Gly
Ser Gly Ala Gly Ala Gly Ser Ala Leu Ser Tyr Pro 850
855 860Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly865 870 875
880Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val
885 890 895Gly Val Pro Gly Val
Gly Val Pro Ala Leu Ser Tyr Pro Gly Ala Gly 900
905 910Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly
Ala Gly Ser Gly 915 920 925Ala Gly
Ala Gly Ser Ala Leu Ser Tyr Pro Gly Val Gly Val Pro Gly 930
935 940Val Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly Lys945 950 955
960Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
965 970 975Val Pro Ala Leu
Ser Tyr Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly 980
985 990Ala Gly Ser Gly Ala Gly Ala Met Asp Pro Gly
Arg Tyr Gln Asp Leu 995 1000
1005Arg Ser His His His His His His 1010
1015333051DNAUnknownDNA encoding SELP 47K-P4 33atggatcccg tcgttttaca
acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat 60cgccttgcag cacatccccc
tttcgccagc gatccgatgg gagcgggtgc cggttctggt 120gcaggcgcgg gctctgcgct
gagctatccg ggtgttggag tgccaggtgt cggtgttccg 180ggtgtaggcg ttccgggagt
tggtgtacct ggaaaaggtg ttccgggggt aggtgtgccg 240ggcgttggag taccaggtgt
aggcgtcccg gcgctgagct atccgggagc gggtgctggt 300agcggcgcag gcgcgggctc
tggagcgggt gccggttctg gtgcaggcgc gggctctgcg 360ctgagctatc cgggtgttgg
agtgccaggt gtcggtgttc cgggtgtagg cgttccggga 420gttggtgtac ctggaaaagg
tgttccgggg gtaggtgtgc cgggcgttgg agtaccaggt 480gtaggcgtcc cggcgctgag
ctatccggga gcgggtgctg gtagcggcgc aggcgcgggc 540tctggagcgg gtgccggttc
tggtgcaggc gcgggctctg cgctgagcta tccgggtgtt 600ggagtgccag gtgtcggtgt
tccgggtgta ggcgttccgg gagttggtgt acctggaaaa 660ggtgttccgg gggtaggtgt
gccgggcgtt ggagtaccag gtgtaggcgt cccggcgctg 720agctatccgg gagcgggtgc
tggtagcggc gcaggcgcgg gctctggagc gggtgccggt 780tctggtgcag gcgcgggctc
tgcgctgagc tatccgggtg ttggagtgcc aggtgtcggt 840gttccgggtg taggcgttcc
gggagttggt gtacctggaa aaggtgttcc gggggtaggt 900gtgccgggcg ttggagtacc
aggtgtaggc gtcccggcgc tgagctatcc gggagcgggt 960gctggtagcg gcgcaggcgc
gggctctgga gcgggtgccg gttctggtgc aggcgcgggc 1020tctgcgctga gctatccggg
tgttggagtg ccaggtgtcg gtgttccggg tgtaggcgtt 1080ccgggagttg gtgtacctgg
aaaaggtgtt ccgggggtag gtgtgccggg cgttggagta 1140ccaggtgtag gcgtcccggc
gctgagctat ccgggagcgg gtgctggtag cggcgcaggc 1200gcgggctctg gagcgggtgc
cggttctggt gcaggcgcgg gctctgcgct gagctatccg 1260ggtgttggag tgccaggtgt
cggtgttccg ggtgtaggcg ttccgggagt tggtgtacct 1320ggaaaaggtg ttccgggggt
aggtgtgccg ggcgttggag taccaggtgt aggcgtcccg 1380gcgctgagct atccgggagc
gggtgctggt agcggcgcag gcgcgggctc tggagcgggt 1440gccggttctg gtgcaggcgc
gggctctgcg ctgagctatc cgggtgttgg agtgccaggt 1500gtcggtgttc cgggtgtagg
cgttccggga gttggtgtac ctggaaaagg tgttccgggg 1560gtaggtgtgc cgggcgttgg
agtaccaggt gtaggcgtcc cggcgctgag ctatccggga 1620gcgggtgctg gtagcggcgc
aggcgcgggc tctggagcgg gtgccggttc tggtgcaggc 1680gcgggctctg cgctgagcta
tccgggtgtt ggagtgccag gtgtcggtgt tccgggtgta 1740ggcgttccgg gagttggtgt
acctggaaaa ggtgttccgg gggtaggtgt gccgggcgtt 1800ggagtaccag gtgtaggcgt
cccggcgctg agctatccgg gagcgggtgc tggtagcggc 1860gcaggcgcgg gctctggagc
gggtgccggt tctggtgcag gcgcgggctc tgcgctgagc 1920tatccgggtg ttggagtgcc
aggtgtcggt gttccgggtg taggcgttcc gggagttggt 1980gtacctggaa aaggtgttcc
gggggtaggt gtgccgggcg ttggagtacc aggtgtaggc 2040gtcccggcgc tgagctatcc
gggagcgggt gctggtagcg gcgcaggcgc gggctctgga 2100gcgggtgccg gttctggtgc
aggcgcgggc tctgcgctga gctatccggg tgttggagtg 2160ccaggtgtcg gtgttccggg
tgtaggcgtt ccgggagttg gtgtacctgg aaaaggtgtt 2220ccgggggtag gtgtgccggg
cgttggagta ccaggtgtag gcgtcccggc gctgagctat 2280ccgggagcgg gtgctggtag
cggcgcaggc gcgggctctg gagcgggtgc cggttctggt 2340gcaggcgcgg gctctgcgct
gagctatccg ggtgttggag tgccaggtgt cggtgttccg 2400ggtgtaggcg ttccgggagt
tggtgtacct ggaaaaggtg ttccgggggt aggtgtgccg 2460ggcgttggag taccaggtgt
aggcgtcccg gcgctgagct atccgggagc gggtgctggt 2520agcggcgcag gcgcgggctc
tggagcgggt gccggttctg gtgcaggcgc gggctctgcg 2580ctgagctatc cgggtgttgg
agtgccaggt gtcggtgttc cgggtgtagg cgttccggga 2640gttggtgtac ctggaaaagg
tgttccgggg gtaggtgtgc cgggcgttgg agtaccaggt 2700gtaggcgtcc cggcgctgag
ctatccggga gcgggtgctg gtagcggcgc aggcgcgggc 2760tctggagcgg gtgccggttc
tggtgcaggc gcgggctctg cgctgagcta tccgggtgtt 2820ggagtgccag gtgtcggtgt
tccgggtgta ggcgttccgg gagttggtgt acctggaaaa 2880ggtgttccgg gggtaggtgt
gccgggcgtt ggagtaccag gtgtaggcgt cccggcgctg 2940agctatccgg gagcgggtgc
tggtagcggc gcaggcgcgg gctctggagc gggtgccatg 3000gacccgggtc gatatcagga
tcttagatct catcaccatc accatcacta a
3051341063PRTUnknownsilk-elastin repeat sequence protein copolymer 34Met
Asp Pro Val Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val1
5 10 15Thr Gln Leu Asn Arg Leu Ala
Ala His Pro Pro Phe Ala Ser Asp Pro 20 25
30Met Gly Ala His Gly Pro Ala Gly Pro Lys Gly Ala His Gly
Pro Ala 35 40 45Gly Pro Lys Gly
Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly 50 55
60Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro
Gly Gly Ala65 70 75
80Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly
85 90 95Gly Ala Gln Gly Pro Ala
Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly 100
105 110Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly
Ala Gln Gly Pro 115 120 125Ala Gly
Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln 130
135 140Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro
Ala Gly Pro Gly Gly145 150 155
160Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro
165 170 175Gly Gly Ala Gln
Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala 180
185 190Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro
Gly Gly Ala Gln Gly 195 200 205Pro
Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala 210
215 220Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln
Gly Pro Ala Gly Pro Gly225 230 235
240Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala
Gly 245 250 255Pro Gly Gly
Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala His Gly Pro 260
265 270Ala Gly Pro Lys Gly Ala His Gly Pro Ala
Gly Pro Lys Gly Ala His 275 280
285Gly Pro Ala Gly Pro Lys Gly Ala His Gly Pro Ala Gly Pro Lys Gly 290
295 300Ala Gln Gly Pro Ala Gly Pro Gly
Gly Ala Gln Gly Pro Ala Gly Pro305 310
315 320Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala
Gln Gly Pro Ala 325 330
335Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly
340 345 350Pro Ala Gly Pro Gly Gly
Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala 355 360
365Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly
Pro Gly 370 375 380Gly Ala Gln Gly Pro
Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly385 390
395 400Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro
Gly Gly Ala Gln Gly Pro 405 410
415Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln
420 425 430Gly Pro Ala Gly Pro
Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly 435
440 445Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly
Pro Ala Gly Pro 450 455 460Gly Gly Ala
Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala465
470 475 480Gly Pro Gly Gly Ala Gln Gly
Pro Ala Gly Pro Gly Gly Ala Gln Gly 485
490 495Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly
Pro Gly Gly Ala 500 505 510Gln
Gly Pro Ala Gly Pro Gly Gly Ala His Gly Pro Ala Gly Pro Lys 515
520 525Gly Ala His Gly Pro Ala Gly Pro Lys
Gly Ala His Gly Pro Ala Gly 530 535
540Pro Lys Gly Ala His Gly Pro Ala Gly Pro Lys Gly Ala Gln Gly Pro545
550 555 560Ala Gly Pro Gly
Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln 565
570 575Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly
Pro Ala Gly Pro Gly Gly 580 585
590Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro
595 600 605Gly Gly Ala Gln Gly Pro Ala
Gly Pro Gly Gly Ala Gln Gly Pro Ala 610 615
620Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln
Gly625 630 635 640Pro Ala
Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala
645 650 655Gln Gly Pro Ala Gly Pro Gly
Gly Ala Gln Gly Pro Ala Gly Pro Gly 660 665
670Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro
Ala Gly 675 680 685Pro Gly Gly Ala
Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro 690
695 700Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro
Gly Gly Ala Gln705 710 715
720Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly
725 730 735Ala Gln Gly Pro Ala
Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro 740
745 750Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala
Gln Gly Pro Ala 755 760 765Gly Pro
Gly Gly Ala His Gly Pro Ala Gly Pro Lys Gly Ala His Gly 770
775 780Pro Ala Gly Pro Lys Gly Ala His Gly Pro Ala
Gly Pro Lys Gly Ala785 790 795
800His Gly Pro Ala Gly Pro Lys Gly Ala Gln Gly Pro Ala Gly Pro Gly
805 810 815Gly Ala Gln Gly
Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly 820
825 830Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly
Gly Ala Gln Gly Pro 835 840 845Ala
Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln 850
855 860Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly
Pro Ala Gly Pro Gly Gly865 870 875
880Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly
Pro 885 890 895Gly Gly Ala
Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala 900
905 910Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly
Pro Gly Gly Ala Gln Gly 915 920
925Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala 930
935 940Gln Gly Pro Ala Gly Pro Gly Gly
Ala Gln Gly Pro Ala Gly Pro Gly945 950
955 960Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln
Gly Pro Ala Gly 965 970
975Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro
980 985 990Ala Gly Pro Gly Gly Ala
Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln 995 1000
1005Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro
Gly Gly 1010 1015 1020Ala His Gly Pro
Ala Gly Pro Lys Gly Ala His Gly Pro Ala Gly Pro1025 1030
1035 1040Lys Met Asp Pro Gly Arg Tyr Gln Leu
Ser Ala Gly Arg Tyr His Tyr 1045 1050
1055Gln Leu Val Trp Cys Gln Lys
1060353024DNAUnknownDNA encoding silk-elastin repeat sequence
35ggcgcgcatg gcccggcggg cccgaaaggc gcgcatggcc cggcgggccc gaaaggcgcg
60cagggcccgg cgggcccggg cggcgcgcag ggcccggcgg gcccgggcgg cgcgcagggc
120ccggcgggcc cgggcggcgc gcagggcccg gcgggcccgg gcggcgcgca gggcccggcg
180ggcccgggcg gcgcgcaggg cccggcgggc ccgggcggcg cgcagggccc ggcgggcccg
240ggcggcgcgc agggcccggc gggcccgggc ggcgcgcagg gcccggcggg cccgggcggc
300gcgcagggcc cggcgggccc gggcggcgcg cagggcccgg cgggcccggg cggcgcgcag
360ggcccggcgg gcccgggcgg cgcgcagggc ccggcgggcc cgggcggcgc gcagggcccg
420gcgggcccgg gcggcgcgca gggcccggcg ggcccgggcg gcgcgcaggg cccggcgggc
480ccgggcggcg cgcagggccc ggcgggcccg ggcggcgcgc agggcccggc gggcccgggc
540ggcgcgcagg gcccggcggg cccgggcggc gcgcagggcc cggcgggccc gggcggcgcg
600cagggcccgg cgggcccggg cggcgcgcag ggcccggcgg gcccgggcgg cgcgcagggc
660ccggcgggcc cgggcggcgc gcagggcccg gcgggcccgg gcggcgcgca tggcccggcg
720ggcccgaaag gcgcgcatgg cccggcgggc ccgaaaggcg cgcatggccc ggcgggcccg
780aaaggcgcgc atggcccggc gggcccgaaa ggcgcgcagg gcccggcggg cccgggcggc
840gcgcagggcc cggcgggccc gggcggcgcg cagggcccgg cgggcccggg cggcgcgcag
900ggcccggcgg gcccgggcgg cgcgcagggc ccggcgggcc cgggcggcgc gcagggcccg
960gcgggcccgg gcggcgcgca gggcccggcg ggcccgggcg gcgcgcaggg cccggcgggc
1020ccgggcggcg cgcagggccc ggcgggcccg ggcggcgcgc agggcccggc gggcccgggc
1080ggcgcgcagg gcccggcggg cccgggcggc gcgcagggcc cggcgggccc gggcggcgcg
1140cagggcccgg cgggcccggg cggcgcgcag ggcccggcgg gcccgggcgg cgcgcagggc
1200ccggcgggcc cgggcggcgc gcagggcccg gcgggcccgg gcggcgcgca gggcccggcg
1260ggcccgggcg gcgcgcaggg cccggcgggc ccgggcggcg cgcagggccc ggcgggcccg
1320ggcggcgcgc agggcccggc gggcccgggc ggcgcgcagg gcccggcggg cccgggcggc
1380gcgcagggcc cggcgggccc gggcggcgcg cagggcccgg cgggcccggg cggcgcgcag
1440ggcccggcgg gcccgggcgg cgcgcatggc ccggcgggcc cgaaaggcgc gcatggcccg
1500gcgggcccga aaggcgcgca tggcccggcg ggcccgaaag gcgcgcatgg cccggcgggc
1560ccgaaaggcg cgcagggccc ggcgggcccg ggcggcgcgc agggcccggc gggcccgggc
1620ggcgcgcagg gcccggcggg cccgggcggc gcgcagggcc cggcgggccc gggcggcgcg
1680cagggcccgg cgggcccggg cggcgcgcag ggcccggcgg gcccgggcgg cgcgcagggc
1740ccggcgggcc cgggcggcgc gcagggcccg gcgggcccgg gcggcgcgca gggcccggcg
1800ggcccgggcg gcgcgcaggg cccggcgggc ccgggcggcg cgcagggccc ggcgggcccg
1860ggcggcgcgc agggcccggc gggcccgggc ggcgcgcagg gcccggcggg cccgggcggc
1920gcgcagggcc cggcgggccc gggcggcgcg cagggcccgg cgggcccggg cggcgcgcag
1980ggcccggcgg gcccgggcgg cgcgcagggc ccggcgggcc cgggcggcgc gcagggcccg
2040gcgggcccgg gcggcgcgca gggcccggcg ggcccgggcg gcgcgcaggg cccggcgggc
2100ccgggcggcg cgcagggccc ggcgggcccg ggcggcgcgc agggcccggc gggcccgggc
2160ggcgcgcagg gcccggcggg cccgggcggc gcgcagggcc cggcgggccc gggcggcgcg
2220catggcccgg cgggcccgaa aggcgcgcat ggcccggcgg gcccgaaagg cgcgcatggc
2280ccggcgggcc cgaaaggcgc gcatggcccg gcgggcccga aaggcgcgca gggcccggcg
2340ggcccgggcg gcgcgcaggg cccggcgggc ccgggcggcg cgcagggccc ggcgggcccg
2400ggcggcgcgc agggcccggc gggcccgggc ggcgcgcagg gcccggcggg cccgggcggc
2460gcgcagggcc cggcgggccc gggcggcgcg cagggcccgg cgggcccggg cggcgcgcag
2520ggcccggcgg gcccgggcgg cgcgcagggc ccggcgggcc cgggcggcgc gcagggcccg
2580gcgggcccgg gcggcgcgca gggcccggcg ggcccgggcg gcgcgcaggg cccggcgggc
2640ccgggcggcg cgcagggccc ggcgggcccg ggcggcgcgc agggcccggc gggcccgggc
2700ggcgcgcagg gcccggcggg cccgggcggc gcgcagggcc cggcgggccc gggcggcgcg
2760cagggcccgg cgggcccggg cggcgcgcag ggcccggcgg gcccgggcgg cgcgcagggc
2820ccggcgggcc cgggcggcgc gcagggcccg gcgggcccgg gcggcgcgca gggcccggcg
2880ggcccgggcg gcgcgcaggg cccggcgggc ccgggcggcg cgcagggccc ggcgggcccg
2940ggcggcgcgc agggcccggc gggcccgggc ggcgcgcatg gcccggcggg cccgaaaggc
3000gcgcatggcc cggcgggccc gaaa
3024365PRTArtificial Sequencerepeat sequence 36Ala Leu Ser Tyr Pro1
5371080PRTArtificial Sequencesynthetic collagen-like peptide
37Gly Ala His Gly Pro Ala Gly Pro Lys Gly Ala His Gly Pro Ala Gly1
5 10 15Pro Lys Gly Ala His Gly
Pro Ala Gly Pro Lys Gly Ala His Gly Pro 20 25
30Ala Gly Pro Lys Gly Ala Gln Gly Pro Ala Gly Pro Gly
Gly Ala Gln 35 40 45Gly Pro Ala
Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly 50
55 60Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly
Pro Ala Gly Pro65 70 75
80Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala
85 90 95Gly Pro Gly Gly Ala Gln
Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly 100
105 110Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly
Pro Gly Gly Ala 115 120 125Gln Gly
Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly 130
135 140Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala
Gln Gly Pro Ala Gly145 150 155
160Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro
165 170 175Ala Gly Pro Gly
Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln 180
185 190Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro
Ala Gly Pro Gly Gly 195 200 205Ala
Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro 210
215 220Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly
Gly Ala Gln Gly Pro Ala225 230 235
240Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala His
Gly 245 250 255Pro Ala Gly
Pro Lys Gly Ala His Gly Pro Ala Gly Pro Lys Gly Ala 260
265 270His Gly Pro Ala Gly Pro Lys Gly Ala His
Gly Pro Ala Gly Pro Lys 275 280
285Gly Ala His Gly Pro Ala Gly Pro Lys Gly Ala His Gly Pro Ala Gly 290
295 300Pro Lys Gly Ala Gln Gly Pro Ala
Gly Pro Gly Gly Ala Gln Gly Pro305 310
315 320Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro
Gly Gly Ala Gln 325 330
335Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly
340 345 350Ala Gln Gly Pro Ala Gly
Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro 355 360
365Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly
Pro Ala 370 375 380Gly Pro Gly Gly Ala
Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly385 390
395 400Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro
Ala Gly Pro Gly Gly Ala 405 410
415Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly
420 425 430Gly Ala Gln Gly Pro
Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly 435
440 445Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly
Ala Gln Gly Pro 450 455 460Ala Gly Pro
Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln465
470 475 480Gly Pro Ala Gly Pro Gly Gly
Ala Gln Gly Pro Ala Gly Pro Gly Gly 485
490 495Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly
Pro Ala Gly Pro 500 505 510Gly
Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala His Gly Pro Ala 515
520 525Gly Pro Lys Gly Ala His Gly Pro Ala
Gly Pro Lys Gly Ala His Gly 530 535
540Pro Ala Gly Pro Lys Gly Ala His Gly Pro Ala Gly Pro Lys Gly Ala545
550 555 560His Gly Pro Ala
Gly Pro Lys Gly Ala His Gly Pro Ala Gly Pro Lys 565
570 575Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly
Ala Gln Gly Pro Ala Gly 580 585
590Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro
595 600 605Ala Gly Pro Gly Gly Ala Gln
Gly Pro Ala Gly Pro Gly Gly Ala Gln 610 615
620Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly
Gly625 630 635 640Ala Gln
Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro
645 650 655Gly Gly Ala Gln Gly Pro Ala
Gly Pro Gly Gly Ala Gln Gly Pro Ala 660 665
670Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala
Gln Gly 675 680 685Pro Ala Gly Pro
Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala 690
695 700Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro
Ala Gly Pro Gly705 710 715
720Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly
725 730 735Pro Gly Gly Ala Gln
Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro 740
745 750Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro
Gly Gly Ala Gln 755 760 765Gly Pro
Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly 770
775 780Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala His
Gly Pro Ala Gly Pro785 790 795
800Lys Gly Ala His Gly Pro Ala Gly Pro Lys Gly Ala His Gly Pro Ala
805 810 815Gly Pro Lys Gly
Ala His Gly Pro Ala Gly Pro Lys Gly Ala His Gly 820
825 830Pro Ala Gly Pro Lys Gly Ala His Gly Pro Ala
Gly Pro Lys Gly Ala 835 840 845Gln
Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly 850
855 860Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly
Ala Gln Gly Pro Ala Gly865 870 875
880Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly
Pro 885 890 895Ala Gly Pro
Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln 900
905 910Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly
Pro Ala Gly Pro Gly Gly 915 920
925Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro 930
935 940Gly Gly Ala Gln Gly Pro Ala Gly
Pro Gly Gly Ala Gln Gly Pro Ala945 950
955 960Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly
Gly Ala Gln Gly 965 970
975Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala
980 985 990Gln Gly Pro Ala Gly Pro
Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly 995 1000
1005Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro
Ala Gly 1010 1015 1020Pro Gly Gly Ala
Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro1025 1030
1035 1040Ala Gly Pro Gly Gly Ala Gln Gly Pro
Ala Gly Pro Gly Gly Ala Gln 1045 1050
1055Gly Pro Ala Gly Pro Gly Gly Ala His Gly Pro Ala Gly Pro Lys
Gly 1060 1065 1070Ala His Gly
Pro Ala Gly Pro Lys 1075 1080
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