Patent application title: Long Acting Human Interferon Analogs
Inventors:
Yan Fu (New York, NY, US)
Zailin Yu (New York, NY, US)
IPC8 Class: AA61K3821FI
USPC Class:
424 854
Class name: Drug, bio-affecting and body treating compositions lymphokine interferon
Publication date: 2009-11-12
Patent application number: 20090280085
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Patent application title: Long Acting Human Interferon Analogs
Inventors:
Yan Fu
Zailin Yu
Agents:
YI LI
Assignees:
Origin: MIAMI, FL US
IPC8 Class: AA61K3821FI
USPC Class:
424 854
Patent application number: 20090280085
Abstract:
Compositions, kits and methods are provided for Interferon analogs in
order to promote general health or for therapeutic treatment of diseases.
Human interferon analogs are made by fusion of interferon with human
serum albumin. The bio-assay shows that the interferon analogs with the
same cell protection against viral attack have 3-10 times longer acting
function than interferon in vivo. These novel long acting interferon
analogs can be used in treatment of patients with viral infection, such
as SARS virus, HIV, HCV, HBV, or HAV, and the cancer diseases, such as
leukemia and malignant melanoma. They also have a 3-5 times longer
shelf-life compared with interferon.Claims:
1. A recombinant protein comprising (a) the amino acid sequence of SEQ ID
NO: 6; or (b) the amino acid sequence encoded by the polynucleotide of
SEQ ID NO: 5.
2. The recombinant protein of claim 1, wherein said protein is a fusion protein of human serum albumin (HSA) and human interferon-.beta. (IFN-.beta.).
3. The recombinant protein of claim 2, wherein said fusion protein has a plasma half-life that is longer than that of IFN-.beta. alone when administered in vivo.
4. The recombinant protein of claim 2, wherein said fusion protein has a shelf-life that is longer than that of IFN-.beta. alone when stored under a same condition.
5. The recombinant protein of claim 2, wherein said fusion protein binds to an antibody of human albumin.
6. The recombinant protein of claim 2, wherein said fusion protein is recombinantly produced in a host cell selected from the group consisting of mammalian and yeast cells.
7. The recombinant protein of claim 6, wherein said yeast cells are selected from the group consisting of Saccharomyces, Hansenula, Canadida, Pichia, Kluyveromyces, Torulaspora, and Schinosaccharomyces.
8. The recombinant protein of claim 7, wherein said Pichia yeast cells are Pichia pastoris cells.
9. The recombinant protein of claim 8, wherein said host cell contains a recombinant vector comprising the polynucleotide of SEQ ID NO: 5.
10. A composition comprising the recombinant protein of claim 1.
11. The composition of claim 10 further comprising a second human serum albumin-interferon fusion protein.
12. The composition of claim 11, wherein said second human serum albumin-interferon fusion protein comprises human serum albumin-interferon-.alpha. fusion protein, human serum albumin-interferon-.gamma. fusion protein, or human serum albumin-interferon-.omega. fusion protein.
13. A kit comprising the recombinant protein of claim 1 and a second human serum albumin-interferon fusion protein.
14. The kit of claim 13, wherein said second human serum albumin-interferon fusion protein comprises human serum albumin-interferon-.alpha. fusion protein, human serum albumin-interferon-.gamma. fusion protein, or human serum albumin-interferon-.omega. fusion protein.
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001]This application is a continuation of patent application Ser. No. 10/851,666, filed May 24, 2004, which claims the benefit under 35 USC 119(e) of provisional application Ser. No. 60/483,984, filed Jun. 30, 2003. This application is also a continuation-in-part of patent application Ser. No. 10/609,346, filed Jun. 26, 2003, now U.S. Pat. No. 7,244,833 B2, which claims the benefit under 35 USC 119(e) of provisional application Ser. No. 60/392,948, filed Jul. 1, 2002. All parent applications are hereby incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002]1. Field of the Invention
[0003]This invention relates to the manufacture and use of recombinant albumin fusion proteins to make human interferon analogs. The novel interferon analogs have the same functions with interferon in bio-assays, in vitro or in vivo. These long acting recombinant interferon analogs that are particularly expressed in yeast can largely improve interferon's therapeutic function.
[0004]2. Description of Related Art
1. Albumin
[0005]Albumin is a soluble, monomeric protein that comprises about one-half of the blood serum protein. Albumin functions primarily as a carrier protein for steroids, fatty acids, and thyroid hormones and plays a role in stabilizing extracellular fluid volume. Mutations in this gene on chromosome 4 result in various anomalous proteins. Albumin is a globular un-glycosylated serum protein of molecular weight 65,000. The human albumin gene is 16,961 nucleotides long from the putative `cap` site to the first poly(A) addition site. It splits into 15 exons which are symmetrically placed within the 3 domains that are thought to have arisen by triplication of a single primordial domain. Albumin is synthesized in the liver as pre-pro-albumin which has an N-terminal peptide that is removed before the nascent protein is released from the rough endoplasmic reticulum. The product, proalbumin, is in turn cleaved in the Golgi vesicles to produce the secreted albumin. HSA has 35 cysteins; in blood this protein monomer has 17-disulfide linkage (Brown, J. R. "Albumin structure, Function, and Uses" Pergamon, New York, 1977). HSA is misfolded when produced intracellularly in yeast without its amino terminal secretion peptide sequence. This conclusion is based on its insolubility, loss of great than 90% of its antigenicity (as compared to human-derived HSA), and formation of large protein aggregates. At present albumin for clinical use is produced by extraction from human blood. The production of recombinant albumin in microorganisms has been disclosed in EP 330 451 and EP 361 991.
[0006]Albumin is a stable plasma transporter function provided by any albumin variant and in particular by human albumin. HSA is highly polymorphic and more than 30 different genetic alleles have been reported (Weikamp L, R, et al., Ann. Hum. Genet., 37 219-226, 1973). The albumin molecule, whose three-dimensional structure has been characterized by X-ray diffraction (Carter D. C. et al., Science 244, 1195-1198, 1989), was chosen to provide the stable transporter function because it is the most abundant plasma protein (40 g per liter in human), it has a high plasma half-life (14-20 days in human, Waldmann T. A., in "Albumin Structure, Function and Uses", Rosenoer V. M. et al (eds), Pergamon Press, Oxford, 255-275, 1977), and above all it has the advantage of being devoid of enzymatic function, thus permitting its therapeutic utilization at high dose.
2. Interferons
[0007]Interferons are a heterogeneous family of multifunctional cytokines whose first demonstrated biological activity was the induction of cellular resistance to virus infection. Antiviral activity of interferon was the only recognized biological function of the interferons for many years. Today interferons are found many other bio-functions. Interferon's actions on cell growth and differentiation and their many immunoregulatory activities are probably of greater fundamental biological significance.
[0008]Two very distinct families of proteins are counted among the interferons. The IFN-α/β "superfamily" (also called type I IFN) encompasses a group of structurally related genes and proteins that are further subdivided into the subfamilies IFN-αI IFN-αII, and IFN-β. The second "family" consists of a single gene encoding a single protein termed IFN-γ (also called type II IFN or immune IFN). It should be made clear at the outset that IFN-γ is structurally unrelated to the members of the IFN-α/β superfamily. The reasons for discussing IFN-α/β and IFN-γ together are largely historical. Interferon was first described by Isaacs and Lindenmann (1957) as a product of virus-infected cells capable of inducing resistance to infection with homologous or heterologous viruses. A functionally related virus inhibitory protein (today termed IFN-γ) was described by Wheelock (1965) as an "Interferon-like" substance produced by mitogen-activated T-lymphocytes. For many years the only properties that made it possible to distinguish IFN-γ from the other interferons were its lack of stability at Ph 2 (Wheelcok 1965) and distinct antigenic specificity (Youngner and Salvin 1973). Only when the sequences of the proteins and genes of the major interferons were revealed in the early 1980s did it become clear what the relationship of the different interferons is to each other. People recognize now that IFN-γ is primarily an immunoregulatory cytokine whereas the potential actions of IFN-α/β extend to a broader variety of cells and tissues.
[0009]Members of the IFN-α/β superfamily represent the classical interferons. The first clear indication of the heterogeneity of the type I interferon proteins came from studies showing that interferons derived from human leukocytes and fibroblasts are antigenically distinct (Havell et al. 1975). Eventually leukocyte and fibroblast interferons were designated IFN-α and -β, respectively (COMMITTEE ON INTERFERON NOMENCLATURE 1980). Most of the information on interferon structure has been derived from gene cloning studies. At least 24 nonallelic human IFN-α genes and pseudogenes have been identified. They can be divided into two distinct subfamilies, termed IFN-αI and -αII (Weissmann and Weber 1986). The IFN-αI subfamily potentially functional genes and several pseudogenes. The IFN-all subfamily is known to comprise only one functional gene and five or six nonallelic pseudogenes. IFN-αI genes encode mature proteins consisting of 165-166 amino acids; IFN-αII gene encodes a mature protein 172 amino acids long. All of the genes encode N-terminal secretive signal peptide presequences (generally 23 residues long) which are removed by proteolytic cleavage before the release of the mature interferon molecule from the cell. While it is clear that a high degree of homology is found among all human IFN-α genes and proteins, the IFN-αII sequences have diverged significantly from the -αI sequences, warranting their classification into a separate subfamily (Capon et al. 1985). In fact, it has been suggested that the IFN-αII subfamily be named IFN-ω (Adolf 1987).
[0010]IFN-α forms vary in molecular mass between 19 and 26 kDa and are produced by monocytes/macrophages, lymphoblastoid cells, fibroblasts, and a number of different cell types following induction by viruses, nucleic acids, glucocorticoid hormones, and low-molecular weight substances. The effects of IFN-α are wide ranging and include potent anti-viral and anti-parasitic activity. In addition, IFN-α has anti-proliferative effects on certain tumor cells. Human IFN-α species lack potential N-glycosylation sites and most members of the IFN-α subfamilies in their native state are not glycosylated (Pestka 1983). Several natural human IFN-α proteins have been purified to homogeneity. They were shown to range in their apparent molecular weights from 16000 to 21000 (Rubinstein et al. 1981). The reason for these large differences in the apparent molecular weights has not been fully explained.
[0011]A single gene for human IFN-β encodes a 166-residue-long mature protein. Homology between IFN-β and members of the IFN-αI subfamily is about 25-30% at the amino acid level and about 45% in the coding sequences at the nucleotide level (Taniguchi et al. 1980). In addition, there is also extensive homology in the 5'nucleotide flanking regions which contain transcriptional promoter and enhancer sequences, reflecting the fact that IFN-α and -β genes are often coordinately induced (Degrave et al. 1981).
[0012]Interferons represent an important class of biopharmaceutical products, which have a proven track record in the treatment of a variety of medical conditions, including the treatment of certain autoimmune diseases, the treatment of particular cancers, and the enhancement of the immune response against infectious agents. To date, five types of interferons have been found in humans: interferon-alpha, interferon-beta, interferon-gamma, interferon-omega and a new form of human and murine interferon, "interferon-ε," which have applications in diagnosis and therapy.
[0013]Interferon is used for treatment of Hepatitis C, B, and broad range of cancers, such as chronic myelogenous leukemia. Hepatitis C is an inflammation of the liver caused by hepatitis C virus infection. The HCV is most common chronic blood-borne disease in China (almost 80 millions HCV carrier) and USA (almost 4 millions HCV carriers), which causes 1 million people death worldwide per year. Chronic hepatitis B is an inflammation of the liver caused by HBV. The HBV infection can be developed into liver cancer and cirrhosis. 500 million people are infected by HBV in worldwide.
[0014]Production of IFN-α/β during virus infections is generally beneficial as it serves to limit the spread of virus and promote recovery (Gresser et al. 1976). In the past few years several types of interferon preparations have been licensed for clinical use. In the United States E. coli-derived recombinant human IFN-α 2 (IFN-α-2a) and IFN-α A (IFN-α-2b) have been approved for use in the treatment of hairy cell leukemia. IFN-α 2 and IFN-α A are both members of the IFN-α1 subfamily and they differ from each other in a single amino acid in position 23 (Arg in α 2 and Lys in α A). One of the preparations has also been approved for the treatment of condylomata acuminate. Other interferon preparations also have been approved for clinical use in some countries, e.g., a natural mixture of several IFN-α subtypes produced in the Namalwa line of human lymphoblastoid cells or natural human IFN-β produced in cultured fibroblasts. The approved use of these interferon preparations some countries includes chronic active hepatitis B, acute viral encephalitides, and nasopharyngeal carcinoma. A preparation of E. Coli-derived recombinant human IFN-γ has been approved for therapeutic use in rheumatoid arthritis in the German Federal Republic. Approved and experimental therapeutic applications of interferons have been extensively covered in a volume devoted to this topic (Finter and Oldham 1985). Interferon-beta, preferably in low doses, is used for stimulation of erythropoiesis in disorders characterized by lack of maturation of progenitor blood cells to red cells (Michalevicz, U.S. Pat. No. 5,104,653).
[0015]Novel polypeptide produced by E. coli transformed with a newly isolated and characterized human IFN-.alpha and the gene is described. The polypeptide exhibits interferon activities such as antiviral activity, cell growth regulation, and regulation of production of cell-produced substances. Those novel interferon are named as Interferon-α-67, by Innis, in U.S. Pat. No. 5,098,703; Interferon-α 54, in U.S. Pat. No. 4,975,276, and Interferon-α 61, in U.S. Pat. No. 4,973,479.
[0016]Therapeutically synergistic mixtures of purified gamma interferon and purified interleukin-2 are provided for treatment of tumor-bearing hosts. Preferably, the gamma interferon and interleukin-2 are obtained from recombinant cell synthesis (Palladino U.S. Pat. No. 5,082,658).
[0017]The invention provides fusion proteins comprising an N-terminal region derived from an interferon-tau (IFN-τ) polypeptide and a C-terminal region derived from another type I interferon polypeptide, such as IFN-α or IFN-β The fusion proteins exhibit reduced cytotoxicity compared to the corresponding unmodified type I interferons. Johnson, et al. U.S. Pat. No. 6,174,996 is the only patent that mentions how to make an interferon fusion protein.
[0018]A method that comprises administering a PEG12000-IFN alpha conjugate to an individual afflicted with a viral infection susceptible of treatment with interferon alpha, preferably chronic hepatitis C, is disclosed. Glue et al. U.S. Pat. No. 5,908,621 is a patent mentions how to make a long acting or slow release form interferons. Shechter et al., (Proc. Natl. Acad. Sci. USA. 2001 January 30; 98 (3): 1212-1217) reported the method to prolong the half-life of human interferon-α2 in circulation by covalently linked seven moieties of 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) to the amino groups of human interferon-α2.
[0019]There is an invention that features a novel hybrid interferon species that comprises a chain of 161 and/or 162 amino acids. The hybrid is novel not only because its new structure, but also for the reason that the hybrid comprises a shortened or truncated segment of alpha interferon. Hence, an entirely new interferon species which does not occur in nature is reported by Leibowitz et al. in U.S. Pat. No. 4,892,743.
[0020]Chang et al. in U.S. Pat. No. 5,723,125 patent disclosed a hybrid recombinant protein consisting of human interferon, preferably interferon-alpha (IFN-α), and human immunoglobulin Fc fragment, preferably gamma 4 chain. These two protein fragments are joined by a peptide linker comprising the sequence Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser. This method makes an interferon-α fusion protein.
[0021]Kriegler, et al. in U.S. Pat. No. 5,324,655 patent reported a virion expression system for a desired protein packaged in an envelope derived from a retrovirus useful in administering proteins which cross cell membranes in order to serve their function. Preferred virions are those that carry an RNA sequence that encodes cytokines or lymphokines, and includes IL-2, multiple drug resistance protein, and TNF. Particularly disclosed is a DNA construct in which a gene encoding tumor necrosis factor (TNF) is directly linked to DNA encoding a human gamma-interferon signal peptide.
[0022]There are some research paper reported that the combination use of interferons could bring some beneficial to patients such as Trotta in U.S. Pat. No. 5,190,751 patent reported the human leukemia T-cells and B-cells are inhibited from proliferating by treatment with a combination of recombinant human alpha and gamma interferons, either simultaneously or sequentially, and the alpha interferon is preferably recombinant human alfa-2b interferon.
[0023]A common feature for any of these administration modes, however, is rapid inactivation of IFN-α in body fluids and in various tissues (O'Kelly, et al., 1985. Proc. Soc. Exp. Biol. Med. 178, 407-411). This in turn leads to the disappearance of the cytokine from the plasma within several hours after administration (Rostaing, et al., 1998, J. Am. Soc. Nephrol. 9, 2344-2348). Unlike many other administered protein drugs, the major route of IFN-α elimination in vivo takes place in the circulatory system through proteolysis and inactivation by serum proteases. Therefore, long acting of interferon is needed in treatment of patients with viral infection or cancers in clinical trials.
DETAILED DESCRIPTION OF THE INVENTION
[0024]The present invention provides innovative compositions, kits and methods for making long acting Interferon analogs in vivo that promote protection of virus infection and stimulate immune response to enhance general health or treat diseases or undesirable conditions.
[0025]In general, recombinant analog of interferon, fusion proteins of human serum albumin (HSA) and an Interferon, are provided in order to circumvent problems associated with conventional therapy using the Interferon protein itself. Generally, compared with the Interferon protein alone, the inventive Interferon analogs of the present invention possess the following advantages: 1) being capable of stimulating immune response of human body while viral infection happen; 2) allowing a slower release of the HSA-Interferon fusion in the body to maximize the therapeutic effects of the Interferon, and/or 3) reducing potential side effects or toxicity associated with administration of Interferon alone.
[0026]The present invention also provides a method for treating a patient with an Interferon in need thereof. In one embodiment, the method comprises administering a pharmaceutical formulation comprising an analog of Interferon to the patient in a therapeutically effective amount. The formulation may contain any pharmaceutically acceptable excipient and agents that stabilizes the HSA/IFN fusion protein. The formulation may further comprises natural or recombinant human serum albumin and/or another, different HSA/IFN fusion protein.
[0027]In addition, the present invention also provides efficient, cost-effective large scale production of these recombinant proteins in yeast. In particular, fusion proteins of HSA with each of human Interferon-α-2a, Interferon-α-2b, and Interferon-co have been expressed in a yeast strain of Pichia pastoria and shown to have superior stability in storage and in plasma with the same bio-function in cell protection experiments in vitro.
1. HSA/IFN Fusion Proteins
[0028]In one aspect of the invention, isolated polynucleotides are provided that encode fusion proteins formed between HSA and an Interferon, i.e., HSA/IFN fusion. It should be noted that other types of albumin can also be employed to produce a fusion protein with an Interferon of the present invention.
[0029]The Interferon may include any protein that belongs to the family of Interferon. In a particular embodiment, the Interferon is a nature active cytokine produced by a virus infection. Examples of such a Interferon are described in Vilcek (1991) "Interferons", in "Peptide Growth Factors and Their Receptors II", edited by Sporn and Roberts, Spring-Verlag Heidelberg, New York Inc., USA. pp 3-38 which is incorporated herein by reference in its entirety.
[0030]Specific examples of the Interferon include, but are not limited to, Interferon alpha-1 (IFNA-1), alpha-2 (IFNA-2), alpha-4 (IFNA-4), alpha-5 (IFNA-5), alpha-6 (IFNA-6), alpha-7 (IFNA-7), alpha-8 (IFNA-8), alpha-10 (IFNA-10), alpha-12 (IFNA-12), alpha-13 (IFNA-13), alpha-14 (IFNA-14), alpha-16 (IFNA-16), alpha-17 (IFNA-17), alpha-21 (IFNA21); Interferon-beta-1 (IFNB-1), interferon-beta-2 (IFNB-2, also be named as interleukin-6, IL-6); Interferon-lambda-1 (Interleukin-29), Interferon-lambda-2 (Interleukin-28A); and/or Interferon-epsilon.
[0031]Three distinct Interferon analogs have been made and well characterized: HSA-INF-α-2a, HSA-INF-α-2b, HSA-INF-β, HSA-INF-ω, and HSA-INF-γ. Other interferons or interferon family members are made by same techniques.
[0032]The Interferon may be linked directly to the N-terminus or C-terminus of HSA to form an analog. Optionally, there is a peptide linker (L) that links HSA and Interferon to form the fusion proteins HSA-L-IFN, or IFN-L-HSA. The length of peptide is usually between 2-100 aa (preferably between 5-50 aa, and most preferably between 14-30 aa). The peptide linker may be a flexible linker that minimizes steric hindrance imposed by the bulk HA protein on interferon, such as a (G4S)3-4 linker. The linker addition may be good for interferon binds to its receptor. The addition of a linker to the in between of HSA and a therapeutic protein needs more work to validated the damage which may cause to when the fusion protein to be used as a therapeutic treatment on human. Because of the 6 amino acids and up peptides can have own immunity in human body. Preferably, there is no linker in the peptide of a human interferon analog. More preferably, there is no linker in the peptide of a long acting of HSA fusion protein drug.
[0033]The fusion protein may be a secret protein, which binds to a specific antibody of human albumin, and optionally, binds to a specific antibody of the interferon in this fusion protein.
[0034]In one embodiment, an isolated polynucleotide is provided that encodes a human serum albumin-interferon-α fusion protein (HSA-IFN-α-1β). The polynucleotide comprises a nucleotide sequence at least 90% identical to SEQ ID NO. 1 (FIG. 1). Preferably, the polynucleotide comprises a nucleotide sequence at least 95% identical to SEQ ID NO. 1. Preferably, the polynucleotide encodes an amino acid sequence comprising SEQ ID NO. 2 [HSA-IFN-α-1b].
[0035]In one embodiment, an isolated polynucleotide is provided that encodes a human serum albumin-interferon-α-2b fusion protein (HSA-IFN-α-2b). The polynucleotide comprises a nucleotide sequence at least 90% identical to SEQ ID NO. 3. Preferably, the polynucleotide comprises a nucleotide sequence at least 95% identical to SEQ ID NO. 3. Preferably, the polynucleotide encodes an amino acid sequence comprising SEQ ID NO. 4 [HSA-IFN-α-2b].
[0036]In another embodiment, an isolated polynucleotide is provided that encodes a human serum albumin-Interferon-β fusion protein (HSA-IFN-β). The polynucleotide comprises a nucleotide sequence at least 90% identical to SEQ ID NO. 5. Preferably, the polynucleotide comprises a nucleotide sequence at least 95% identical to SEQ ID NO. 5. Preferably, the polynucleotide encodes an amino acid sequence comprising SEQ ID NO. 6. [HSA-IFN-β].
[0037]In yet another embodiment, an isolated polynucleotide is provided that encodes a human serum albumin-Interferon-ω fusion protein (HSA-IFN-ω). The polynucleotide comprises a nucleotide sequence at least 90% identical to SEQ ID NO. 7. Preferably, the polynucleotide comprises a nucleotide sequence at least 95% identical to SEQ ID NO. 7. Preferably, the polynucleotide encodes an amino acid sequence comprising SEQ ID NO. 8 [HSA-IFN-ω].
[0038]In yet another embodiment, an isolated polynucleotide is provided that encodes a human serum albumin-Interferon-γ fusion protein (HSA-IFN-γ). The polynucleotide comprises a nucleotide sequence at least 90% identical to SEQ ID NO. 9. Preferably, the polynucleotide comprises a nucleotide sequence at least 95% identical to SEQ ID NO. 9. Preferably, the polynucleotide encodes an amino acid sequence comprising SEQ ID NO. 10 [HSA-IFN-γ].
[0039]In yet another embodiment, an isolated polynucleotide is provided that encodes a human serum albumin-Interferon fusion protein (HSA-IFN). The polynucleotide comprises a nucleotide sequence at least 90% identical to SEQ ID NO. 11. Preferably, the polynucleotide comprises a nucleotide sequence at least 95% identical to SEQ ID NO. 11. Preferably, the polynucleotide encodes an amino acid sequence comprising SEQ ID NO. 12 [HSA].
[0040]Optionally, the polynucleotide further comprises a nucleotide sequence at least 90% identical to SEQ ID NOs. 13, 15, 17, 19, or 21. Preferably, the polynucleotide further comprises a nucleotide sequence encoding an amino acid sequence comprising SEQ ID NOs. 14, 16, 18, 20, or 22.
[0041]According to the embodiment, the Interferon may be selected from the group consisting, such as, but not limited, Interferon alpha-1 (IFNA-1), alpha-2 (IFNA -2), alpha-4 (IFNA-4), alpha-5 (IFNA-5), alpha-6 (IFNA-6), alpha-7 (IFNA-7), alpha-8 (IFNA-8), alpha-10 (IFNA-10), alpha-12 (IFNA-12), alpha-13 (IFNA-13), alpha-14 (IFNA-14), alpha-16 (IFNA-16), alpha-17 (IFNA-17), alpha-21 (IFNA21); Interferon-beta-1 (IFNB-1), interferon-beta-2 (IFNB-2, also be named as interleukin-6, IL-6); Interferon-lambda-1 (Interleukin-29), Interferon-lambda-2 (Interleukin-28A); and/or Interferon-epsilon.
[0042]The above-described polynucleotide with a sequence having a certain degree of sequence identity, for example at least 95% "identical" to a reference nucleotide sequence encoding a HSA/IFN fusion protein, is intended that the polynucleotide sequence is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the HSA/IFN fusion protein. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
[0043]As a practical matter, whether any particular nucleic acid molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the polynucleotide sequence encoding a HSA/IFN fusion protein can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
[0044]When stored at ambient temperature or a lower temperature, the fusion protein of HSA and IFN may have a shelf-life 2 times longer, preferably 4 times longer, more preferably 6 times, and most preferably 10 times, longer than that of the IFN alone stored under the same condition.
[0045]The present invention involves the utilization of albumin as a vehicle to carry a therapeutic protein such as an IFN in the treatment of certain diseases such as cancers, or people in need of an increased blood cell proliferation in order to increase the blood cell numbers. The fusion protein of the present invention may be administered to a mammal, preferably a human, via a variety of routes, including but not limited to, orally, parenterally, intraperitoneally, intravenously, intraarterially, topically, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery (for example by catheter or stent), subcutaneously, intraadiposally, intraarticularly, or intrathecally. The analogs of Interferon, HSA-IFN, may also be delivered to the host locally (e.g., via stents or catheters) and/or in a timed-release manner. In a particular embodiment, the fusion protein is delivered parenterally via injection.
[0046]When delivered in vivo to an animal, the fusion protein of HSA and IFN, Interferon analogs, may have a plasma half-life 2-10 times longer than that of the IFN alone.
[0047]The HSA/IFN fusion proteins of the present invention may also be administered in combination with a natural or recombinant human albumin, preferably a recombinant one at a therapeutically effective dose and ratio.
[0048]It is believed that after fusion with albumin, the IFN protein can have a longer shelf-life and plasma half-life, which allows cost-effective storage and transportation, as well as reduces amount and/or frequency of drug administration.
[0049]It is believed that other polypeptide form anti-virus or peptide inhibitors of virus entry cell after fusion with albumin, the peptide protein can have a longer shelf-life and plasma half-life, which allows maintaining same bio-function of peptide and gives a long acting therapeutic function. The peptides such as T20 can block the HIV virus entry of HIV targeted cells
2. Expression of Interferon Analogs in Host Organisms
[0050]The polynucleotides encoding the inventive Interferon analogs, HSA/IFN fusion proteins, can be cloned by recombinant techniques into vectors which are introduced to host cells where the fusion proteins can be expressed.
[0051]Generally, host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the polynucleotides encoding HSA/IFN fusion proteins. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
[0052]According to the invention, a recombinant vector is provided that comprises the polynucleotide sequence encoding an HSA/IFN fusion protein. The recombinant vectors can be an expression vector for expressing the Interferon analogs, HSA fusion protein encoded by the nucleic acid, HSA-IFN, HSA-L-IFN, or IFN-L-HSA in a host organism. The host organism includes, but is not limited to, mammalian (e.g., human, monkey, mouse, rabbit, etc.), fish, insect, plant, yeast, and bacterium.
[0053]Expression of the polynucleotide encoding an HSA/IFN fusion protein is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, a tetracycline or tetracycline-like inducible promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the β-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter which controls the polynucleotide encoding an HSA/IFN fusion protein.
[0054]Also according to the invention, a recombinant cell is provided that is capable of expressing comprises the polynucleotide sequence encoding an HSA/IFN fusion protein. The recombinant cell may constitutively or be induced in the presence or absence of an agent to express Interferon analog, HSA fusion protein, encoded by the nucleic acid, HSA-IFN, HSA-L-IFN, or IFN-L-HSA in a host organism. The type of the recombinant cell includes, but is not limited to, mammalian (e.g., human, monkey, mouse, rabbit, etc.), fish, insect, plant, yeast, and bacterial cell.
[0055]In a preferred embodiment, the host organism belongs to a genus of yeast such as Saccharomyces (e.g., S. cerevisiae), Pichia, Kluyveromyces, Hansenula, Torulaspora, and Schinosaccharomyces. In a more preferred embodiment, the host organism is Pichia pastoris. In a particular embodiment, the recombinant vector is a pPICZ A, pPICZ B, or pPICZ C.
[0056]Depending upon the host employed in a recombinant process for producing the fusion proteins, the fusion proteins of the present invention may be glycosylated or non-glycosylated. Preferably, when expressed in a host organism, the fusion protein of HSA and IFN may be glycosylated to substantially the same extent as that when expressed in mammalian cells such as Chinese hamster ovarian (CHO) cells, or as that when expressed in Pichia pastoris.
[0057]As indicated above, the albumin fusion proteins of the present invention are substantially preferably proteomic and can therefore be generated by the techniques of genetic engineering. The preferred way to obtain these fusion proteins is by the culture of cells transformed, transfected, or infected by vectors expressing the fusion protein. In particular, expression vectors capable of transforming yeasts, especially of the genus Pichia, for the secretion of proteins will be used.
[0058]It is particularly advantageous to express the HSA/IFN fusion protein in yeast. Such an expression system allows for production of high quantities of the fusion protein in a mature form, which is secreted into the culture medium, thus facilitating purification.
[0059]The development of yeast genetic engineering has been made possible the expression of heterologous genes and the secretion of their protein products from yeast. The advantages of protein secretion (export) of yeast include, but not limited to, high expression level, soluble protein, corrected folding, easy to scale-up and easy for purification.
[0060]HSA/IFN fusion proteins, the Interferon analogs, can be secreted into the media of yeast via an albumin natural secretion signal. The polypeptide sequence of HSA fusion protein can be preceded by a signal sequence which serves to direct the proteins into the secrete pathway. In a preferred embodiment the prepro-sequence of human albumin is used to secrete the fusion protein out of yeast cells into the culture medium. Other secrete signal peptides, such as the native Saccharomyces cerevisiae α-factor secretion signal, can also be used to make fusion protein of the present invention.
[0061]Yeast-expressed HSA is soluble and appears to have the same disulfide linkages as the human-blood derived counterpart. If used in a large scale production, which may be potentially used in gram amounts in humans, a recombinant HSA will require a close identity with the natural HSA product. Secreting the HSA/IFN fusion protein into the growth media of yeast, which is via prepro-amino-terminal processing (no initiator methionine residue), also circumvents the problems associated with preparing yeast extracts, such as the resistance of yeast cells to lysis. In addition, the purity of the product can be increased by placing the product in an environment in which 0.5-1.0% of total yeast proteins is included and the lacks toxic proteins that would contaminate the product.
[0062]In a preferred embodiment, a particular species of yeast Pichia pastoris is used in the system for expressing HSA/IFN fusions of the present invention. Pichia pastoris was developed into an expression system by scientists at Salk Institute Biotechnology/Industry Association (SIBA) and Phillips Petroleum for high-level expression of recombinant proteins. The techniques related to Pichia are taught in, for example, U.S. Pat. Nos. 4,683,293, 4,808,537, and 4,857,467.
[0063]There are some advantages of using yeast Pichia pastoris to express HSA and HSA fusion proteins than using other systems. Pichia pastoris is a species of yeast genus, Pichia. Pichia has many advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while it is as easy to manipulate as E. coli or Saccharomyces cerevisiae. It is faster, easier, and less expensive to use than other eukaryotic expression systems such as baculovirus or mammalian tissue culture, and generally gives higher expression levels. Pichia has an additional advantage which gives 10-100 fold higher heterogonous protein expression levels. Those features make Pichia a very useful protein expression system.
[0064]Due to the similarity between Pichia and Saccharomyces, many techniques developed for Saccharomyces may be applied to Pichia. These include transformation by complementation, gene disruption, and gene replacement. In addition, the genetic nomenclature used for Sac has been applied to Pichia. For example, histidinol dehydrogenase is encoded by HIS4 gene in both Sac and Pichia. Pichia as a methylotrophic yeast is capable of metabolizing methanol as its sole carbon source. The first step in the metabolism of methanol is oxidation of methanol to formaldehyde using molecular oxygen by the enzyme called alcohol oxidase. In addition to formaldehyde, this reaction also generates hydrogen peroxide. To avoid hydrogen peroxide toxicity, methanol metabolism takes place within a specialized cell organelle, called the peroxisome, which sequesters toxic by-products away from the rest of the cell. Alcohol oxidase has a poor affinity for O2, and Pichia compensates it by generating large amounts of this enzyme. The promoter regulating the production of alcohol oxidase is the one used to drive heterogonous (HSA or HSA fused) protein expression in Pichia.
[0065]Compared with Saccharomyces cerevisiae, Pichia may have an advantage in glycosylation of secrete proteins because it generally does not hyper-glycosylate. Both Saccharomyces and Pichia have a majority of N-linked glycosylation of the high-mannose type; however, the length of the oligosaccharide chains that add post-translation ally to proteins in Pichia (average 8-14 mannose residues per side chain) is much shorter than those in Saccharomyces (50-150 mannose residues). Very little O-linked glycosylation has been observed in Pichia. In addition, Saccharomyces core oligosaccharide has terminal α-1,3 glycan linkages whereas Pichia does not. It is believed that the α-1,3 glycan linkages in glycosylated proteins produced from Saccharomyces are primarily responsible for the hyper-antigenic nature of those proteins that make them particularly unsuitable for therapeutic use. Although not yet proven, this is predicted to be less of a problem for glycoprotein generated in Pichia, because it may resemble the glycoprotein structure of higher eukaryotes. Protein expressed as a secrete form for correctly refolding and easy purification of HSA and HSA fusion proteins.
[0066]Watanabe, et al. (2001) "In vitro and in vivo properties of recombinant human serum albumin from Pichia pastoris purified by a method of short processing time", Pharm Res 2001 December:18(12):1775; and Kobayashi, K et al. (1998) "The development of recombinant human serum albumin" Ther Apher, November:2(4):257-62.
[0067]There are many expression systems available for expressing in Pichia, such as EasySelect® Pichia Expression Kit from Invitrogen, Inc. On this vector, an AOX1 promoter is used to allow methanol-inducible high level expression in Pichia and a Zeocin® resistance as selective market for the recombinants from the transformation. Promoters (transcription initiation region) are very important in expressing fusion proteins in this invention.
[0068]AOX1 gene promoter is very strong in yeast system, especially in Pichia. Two Alcohol Oxidase Proteins are coded in Pichia for alcohol oxidase--AOX1 and AOX2. The AOX1 gene is responsible for the vast majority of alcohol oxidase activity in the cell. Expression of the AOX1 gene is tightly regulated and induced by methanol to very high levels, typically ≧30% of the total soluble protein in cells grown with methanol as the carbon source. The AOX1 gene has been isolated and a plasmid-bone version of the AOX1 promoter is used to drive expression of the gene of interest encoding the desired heterogonous protein (Ellis et al., 1985; Koutz et al., 1989; Tschopp et al., 1987a). While AOX2 is about 97% homologous to AOX1, growth on methanol is much slower than with AOX1. This slow growth on methanol allows isolation of Muts strains (aox1). Except for AOX1 gene promoter, other promoters can also be used to driver HSA fusion gene in yeast. They include the promoter from, but not limited to, PGK1, GAPDH, Gal1, Gal10, CYC1, PHO5, TRP1, ADH1, and ADH2 genes. In this invention, we also disclose a novel method to make recombinant yeast with dual expression cassette insertions at two separated locations.
[0069]The expression plasmid can also take the form of shuttle vectors between a bacterial host such as E. coli, DH5a from GIBCO/Life Science and yeast. The antibiotic Zeocin are used to be a marker for HSA carrier vector in all the examples.
[0070]The expression vector that contains the polynucleotide of HSA or HSA fusion therapeutic protein is introduced into yeast according to the protocols described in the kit from Invitrogen Inc. After being selected from transformed yeast colonies, those cells that express the HSA fusion protein of interest are inoculated into appropriate selective medium and then tested for their capacity to secrete the given fusion protein into the extracellular medium. The harvest of the protein can be conducted during cell growth for continuous cultures, or at the end of the growth phase for batch cultures. The fusion proteins which are the subject of this invention are then further purified from the culture supernatant by methods which take into account the albumin purification methods and pharmacological activities.
[0071]It is noted that other expression systems may also be used to express rHSA and HSA/IFN fusion proteins, including but not limited to, E. coli, B. Subtitis, Saccharomyces, Kluyveromyces, Hansenula, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis, animals, plants, and insect cells.
3. Combination Therapy of Interferon Analogs
[0072]The present invention also provides combinations of different Interferon analogs. The specific combinations of these interferon analogs or nature interferons may be administered to a patient to stimulate multiple types of protection to viral targeted cells or to synergistically enhance proliferation of a particular cell type. In particular, a combination of human albumin fusions with different hematopoietically active cytokines is used to effectively promote proliferation of the multiple blood cells and platelets. By using a combination of HSA/IFN fusion proteins targeting the signal transduction pathways of different types of blood cells, multiple blood functional cell production, such as platelets, erythrocytes and macrophages of white cells, can be increased after administration by just one injection.
[0073]In the present invention, the albumin's plasma transporter function and the therapeutic function of the IFN are integrated into a fusion form. The presence of albumin may confer a superior stability to the IFN by resisting degradation by proteases in the blood circulation, thus significantly prolonging the plasma half life of the IFN. Due to the masking effect of a bulky albumin, different IFNs fused with albumin in the combination may impose less interference with the biological function(s) of each other than a combination of the "naked" IFNs. Furthermore, an IFN fused with albumin may be slowly released in the system over an extensive period of time, thereby reducing the toxicity associated with injection of the IFN alone in abnormally high concentrations in the body. Such a slow release mode of action of the fusion protein combination can significantly reduce the amount and/or frequency of injections of the IFN, thereby further reducing the side effects of IFNs. Such combinations that are particularly useful for stimulating multiple blood cell proliferation after or before the chemo- or radiation therapy of cancer patients who are tolerance for frequent, high dose injection of IFN are seriously compromised.
[0074]According to the present invention, HSA fusion protein with this type of IFN may remove above limitations by slowly releasing the drug into the patient's system. In addition, such fusion proteins may be combined with a relatively higher amount of albumin to further reduce the impact resulted from directly injecting the drug into the blood which causes a strong, adverse reaction of the central nervous system.
[0075]It is also known that "naked" cytokines (i.e., cytokines not fused to another protein such as HSA) are quite unstable when stored and have a short plasma half-life. Clearly, a therapeutic protein with such a weak stability in vivo constitutes a major handicap. In effect, repeated injections of the product, which are costly and inconvenient for patient, or an administration of product by perfusion, become necessary to attain an efficient concentration in plasma. Due to its extended plasma half and enhanced stability, the HSA/IFN fusion proteins of the present invention and their combinations, e.g., HSA fusions with Interferon-α, interferon-β, interferon-ω and interferon-γ, can be used to stimulate the production of antivirus peptides in plasma of humans.
[0076]In one embodiment, HSA/IFN-α fusion may be combined with HSA/IFN-γ fusion and the resulting combination may be administered to a patient with a virus infection to simultaneously stimulate secretion of antiviral peptides. For example, cancer patients may be injected with a combination of HSA/IFN-α and HSA/IFN-γ fusion proteins, before or after, a viral infection to avoid the damages of cells and organs. The Interferon-a will promoter the fight with virus and Interferon-g will fight inhibit the cancer cell proliferation.
[0077]Alternatively, an HSA/IFN fusion may be co-administered with a different HSA/IFN fusion simultaneously or sequentially to a patient in need thereof. This combination therapy may confer synergistic therapeutic effects on the patients. In one embodiment, the method is provided, comprising: administering a first pharmaceutical formulation comprising a first fusion protein of HSA and a first IFN to the patient in a therapeutically effective amount; and administering to the patient a second pharmaceutical formulation comprising a second fusion protein of HSA and a second IFN to the patient in a therapeutically effective amount. Such a combination therapy may confer synergistic therapeutic effects on the patient.
[0078]For example, HSA-IFN-α-2b fusion protein may be administered to the patient first, followed by administration of HSA-IFN-γ, HSA-IFN-ω and/or HSA-IFN-β at therapeutically effective doses and ratios to inhibit cancer cell proliferation of different and to induce antiviral peptide secretion from cells.
[0079]The present invention further provides a kit for use in the combination therapy described above. The kit comprises: a first fusion protein of HSA and a first IFN, and a second fusion protein of HSA and a second IFN. The first and second IFNs may be the same or different. For example, the first IFN is IFN-α-2b and the second IFN is Interferon-γ; the first IFN is Interferon-ω and the second IFN is Interferon-γ; or the first IFN is Interferon-β and the second IFN is Interferon-γ.
[0080]The HSA/IFN fusion proteins and their combinations thereof may be used to treat a wide variety of diseases, including but not limited to, the viral infection, such HAV, HBV, HCV, HPV, SARS virus, and/or HIV infection, tumors, cancers, renal failure, and tissue/organ transplantation. These fusion proteins are preferred not to contain non-human sequences that may elicit adverse immunogenicity in the patient.
[0081]Interferon analogs are including but not limited to Interferon alpha-1 (IFNA-1), alpha-2 (IFNA-2), alpha-4 (IFNA-4), alpha-5 (IFNA-5), alpha-6 (IFNA-6), alpha-7 (IFNA-7), alpha-8 (IFNA-8), alpha-10 (IFNA-10), alpha-12 (IFNA-12), alpha-13 (IFNA-13), alpha-14 (IFNA-14), alpha-16 (IFNA-16), alpha-17 (IFNA-17), alpha-21 (IFNA21); Interferon-beta-1 (IFNB-1), interferon-beta-2 (IFNB-2, also be named as interleukin-6, IL-6); Interferon-lambda-1 (Interleukin-29), Interferon-lambda-2 (Interleukin-28A); and/or Interferon-epsilon.
BRIEF DESCRIPTION OF THE FIGURES
[0082]FIG. 1 shows nucleotide and amino acid sequences of embodiments of Analogs of Interferon, HSA, and examples of individual IFNs.
[0083]FIG. 2 illustrates a plasmid DNA vector contains the HSA sequence and as a backbone vector for making Interferon analogs, HSA-IFN fusion proteins.
[0084]FIG. 3 shows a Western blot detected using mouse monoclonal anti-human serum albumin (Sigma Cat# A6684). Each lane was load with equivalent of 10 μl of culture medium supernatant from yeast after three-day expression. A), HSA (65 Kd); B), Analog IFN-α-2a (84 Kd); C). Analog IFN-β (84 kd); D). Analog IFN-ω (84 kd); E). Control (yeast parent strain culture).
[0085]FIG. 4 shows a Western blot detected using Rabbit polyclonal anti-hIFN-α-2a antibody (Chemicon International Inc., Cat# Ab-218-NA), each lane contains 100 ng proteins. A), human IFN-α-2b (19 kd) expressed by E. coli; B), Analog Interferon-α 84 kd., HSA/IFN-α-2b fusion protein, expressed by yeast.
[0086]FIG. 5 is an Antiviral infection assay for human IFN-α and Analog Interferon-α, HSA/IFN-α-2a fusion protein, in WISH cell with VSV challenges.
[0087]FIG. 6 shows the results of a stability test of Interferon analog proteins under different temperature and its cell viral protection activity. A), 37° C.; B), 50° C.
[0088]FIG. 7 shows the long acting effects in vivo test of analog interferon, HSA-IFNs, in animal plasma, as compared with those when Interferon Analog or IFN were administered. A), 1-24 hrs; B), 1-12 days.
EXAMPLES
1. General Molecular Cloning Techniques
[0089]The classic methods of molecular cloning that include DNA preparative extractions, agarose and polyacrylamide electrophoresis, plasmid DNA purification by column or from gel, DNA fragment ligations, and restriction digestion are described in detail in Maniatis T. et al., "Molecular cloning, a Laboratory Manual", Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y., 1982 and will not be reiterated here.
[0090]Polymerase Chain Reaction (PCR) used through out all the examples is described by Saiki, R. K. et al, Science 230:1350-1354, 1985 and is carried out on a DNA thermal cycler (Perkin Elmer) according to the manufacturer's specification. DNA sequencing was performed by using standard facilities and following the method developed by Sanger et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467, 1977. Oligonucleotides were synthesized by commercial facilities.
[0091]Transformation of E. coli was done by using DH5α competent cells from GIBCO/BRL. Qiagen plasmid DNA purification columns were used in the purification of plasmid DNAs. The transformation of yeast was carried out by electroporation following the instruction provided by the manufacturer or according to the manual of EasySelect® Pichia Expression Kit (Invitrogen Inc). All yeast stains used in the examples are members of the family of Pichia, and in particular, the strain of Pichia pastoris (supplied by Invitrogen).
2. Construction of a Backbone Vector Expressing Human Serum Albumin
[0092]A total RNA isolated from human fetal liver was used in a reverse transcription polymerase chain reaction (RT-PCR) to generate the polynucleotide encoding human serum albumin. Briefly, 5 μg of RNA was reverse transcribed by adding a poly(T)18+N primer and the SuperScript® II RNase H.sup.- reverse transcriptase (GIBCO/BRL) to make the complementary first strand of cDNA. The reaction was incubated at 45° C. for 20 minutes, then at 55° C. for 40 minutes.
[0093]The primers for cloning human serum albumin (HSA) are the following:
TABLE-US-00001 SEQ ID No. 23: 5'-GAATTCATGAAGTGGGTAACCTTTATTTCC-3' and SEQ ID No. 24: 5'-GAATTCTTATAAGCCTAAGGCAGCTTGACTTGC-3'.
[0094]These primers were designed based on the HSA sequence published by GenBank (Access# V00494). Two EcoR I (underline of primers) sites were created at the 5' end and 3' end for sub-cloning into an expression vector. After inactivating the reverse transcriptase at 94° C. for 4 minutes, the DNA encoding of HSA was further amplified by Taq DNA PCR (Perkin Elmer) with 35 cycles of 94° C./30 seconds and 58° C./30 seconds and 72° C./2 minutes 30 second, followed by a 72° C./10 minutes incubation. The PCR product (1842 base pairs) was confirmed by 1% agarose gel electrophoresis. The product was subcloned into a pCR II TA cloning vector from Invitrogen. DNA sequencing confirmed that the plasmid DNA contained an insert whose polynucleotide sequence matches the DNA sequence published in GenBank (Access# V00494). FIG. 1, Seq ID No. 11 is a polynucleotide DNA sequence and Seq ID No 12 is the protein amino acid sequence of human serum albumin.
[0095]After restriction digestion of the PCR product with EcoR I, the gel purified HSA DNA fragment was inserted into the EcoR I site of a pPICZ-A or pGAPZ-A vector (provided by Invitrogen) or a new vector, pYH, modified by Zailin YU. After transformation of bacteria DH5α cells with this vector encoding HSA, a colony was selected from a low salt LB-agar plate contains 25 μg/ml Zeocin. The direction of the insert was confirmed by restriction enzyme double digestion of plasmid DNA by Xho I/Nde I. The constructs were designated as pYZ-HSA (Y: yeast vector; Z: Zeocin resistant) driven by AOX1 or GAP promoter; or pYH-HSA (Y: yeast vector, Histidine resistant) driven by AOX1 or GAP (GAPDH) promoter and its physical maps are shown in FIG. 2.
[0096]There are some advantages associated with the vector constructed above. 1) It confers resistance to the antibiotic Zeocin. Zeocin is isolated from Streptomyces and is structurally related to bleomycin/phleomycin-type antibiotics. Antibiotics in the family of bleomycin/phleomycin are broad spectrum antibiotics that act as strong antibacterial and anti-tumor drugs. They show strong toxicity against bacteria, fungi (including yeast), plants, and mammalian cells. However, Zeocin is not as toxic as bleomycin on fungi. A single antibiotic Zeocin could be used in selecting the recombinants in both bacteria and in yeast. Further, there are multiple cloning sites at the 3' end of HSA for conveniently subcloning an IFN protein in frame to encode a HSA-IFN. 2) A myc epitope sequence and a polyhistidine tag can be fused to the C-terminal of the expressed fusion protein for easy detection and/or purification by using commercially available antibodies against myc or polyhistidine tags. 3) AOX1 promoter or GAP promoter could be used which gives more choice for convenient expression of HSA/IFN. The GAP promoter is a no methanol inducer. By using of GAP promoter than AOX promoter, the industry scale level (1,000 Kg) fermentation would be safer with no use of methanol as an additive to induce the expression. 4) A dual expression cassette (promoter, to be expressed gene and resistant gene) from two vectors could be directly inserted with controlling into same yeast strain to make recombinant yeast for higher expression. Two vectors with promoter and insert, same or not, could be transformed into a yeast strain., pYZ-HSA, will directly insert at AOX1 gene locus with Zercin resistant, using same promoter's pYH-HSA, will directly insert at His gene location with H is selection function. Vectors, pYH and pYZ as backbone vectors, were used in the construction of expression vectors for HSA fusion proteins described in the Example section.
3. Molecular Cloning of Human Interferons
3.1. Molecular Cloning Of Human Interferon-α-1b Gene
[0097]Human Interferon-α-1b was cloned from a total RNA preparation of human white blood cells (monocytes/macrophages and B lymphocytes) by RT-PCR method described in Example 2. The oligonucleotide primers are
TABLE-US-00002 SEQ ID NO. 25: 5'-CATATGTGTGATCTCCCTGAGACCC-3' SEQ ID NO. 26: 5'-GGATCCTTACTTCCTCCTTAATCTTTC-3'
[0098]A polynucleotide having 509 base pairs (bp) was amplified from RT-PCR reaction and subcloned into pCR II TA cloning vector from Invitrogen Inc. DNA sequencing confirmed the reading frame of human Interferon-α-1b. An Nde I restriction enzyme site was created at the 5' end and a Bam HI site at the 3' end (underline). The ATG initiate start codon of Interferon-a was included in this site (underlined in SEQ ID NO. 25). The DNA sequence of human Interferon-α-1b (SEQ ID NO. 13) and its amino acid sequence (SEQ ID NO. 14) are shown in FIG. 1.
3.2. Molecular Cloning Of Human Interferon-α-2a Gene
[0099]Human Interferon-α-2a was cloned from a total RNA preparation of human white blood cells (monocytes/macrophages and B lymphocytes) by RT-PCR method described in Example 2. The oligonucleotide primers are
TABLE-US-00003 SEQ ID NO. 27: 5'-CATATGGCCTTGACCTTTGCTTTAC-3' SEQ ID NO. 28: 5'-GGATCCTCATTCCTTACTTCTTAAAC-3'
[0100]A polynucleotide having 579 base pairs (bp) was amplified from RT-PCR reaction and subcloned into pCR II TA cloning vector from Invitrogen Inc. DNA sequencing confirmed the reading frame of human Interferon-α-2a. An Nde I restriction enzyme site was created at the 5' end and a Bam HI site at the 3' end (underline). The ATG initiate start codon of Interferon-a was included in this site (underlined in SEQ ID NO. 27).
3.3. Molecular Cloning of Human Interferon-α-2b Gene
[0101]Human Interferon-α-2b gene has only one nucleotide different with Interferon-α-2a gene that result gives an amino acid different in position #23 (Arg in interferon-α-2a and Lys in interferon-α-2b). The interferon-α-2b gene was obtained by point mutation from cloned interferon-α-2a by a kit from Stratagene company. A paired mutation primers are used to make one nucleotide change in sequence. They are
TABLE-US-00004 SEQ ID NO. 29: 5'-TGGCACAGATGAGGAAAATCTCTCTTTTCTCCTGC-3', and SEQ ID NO. 30: 5'-CAGGAGAAAAGAGAGATTTTCCTCATCTGTGCCAGC-3'.
[0102]The underlined nucleopeptide is the mutation point, from Interferon-α-2a, AGA (Arg) to Interferon-α-2b, AAA (Lys). The experiment was performed according to the manufacture's instruction. Mutated product in pCR II vector was sequence confirmed. The human Interferon-α-2b gene DNA sequence (SEQ ID NO. 15) and amino acid sequence (SEQ ID NO. 16) are showed in FIG. 1.
3.4. Molecular Cloning Of Human Interferon-β
[0103]Primers used to clone the human Interferon-β gene from a cDNA library of human leukocyte are
TABLE-US-00005 SEQ ID NO. 31: 5'-CATATGACCAACAAGTGTCTCC-3', and SEQ ID NO. 32: 5'-GAATTCTCAGTTTCGGAGGTAACC-3'
[0104]An Nde I site created at 5' end and an EcoR I site at 3' end of Interferon-β were created. The PCR products were gel-purified and subcloned into pCR2.1 TA cloning vectors and DNA sequence was confirmed. The human interferon-β DNA sequence (SEQ ID NO. 17) and the amino acid sequence (SEQ ID NO. 18) are shown in FIG. 1.
3.5. Molecular Cloning Of Human Interferon-ω
[0105]Human interferon-ω was cloned from a total RNA sample prepared from human cDNA Library of Leukocyte (White Blood Cells). The primers were:
TABLE-US-00006 SEQ ID NO. 33: 5'-CATATGGCCCTCCTGTTCCCTCTAC -3', and SEQ ID NO. 34: 5'-GAATTCTCAAGATGAGCCCAGGTCTC-3'
[0106]The PCR products were gel-purified and inserted into pCR2.1 TA cloning vector and sequence confirmed. The human Interferon-ω DNA sequence (SEQ ID NO. 19) and amino acid sequence (SEQ ID NO. 20) are shown in FIG. 1.
3.6. Molecular cloning Of Human Interferon-γ
[0107]Human interferon-γ was cloned from a total RNA sample prepared from human cDNA library of mitogen-activated T-lymphocytes. The primers were:
TABLE-US-00007 SEQ ID NO. 35: 5'-CATATGAAATATACAAGTTATATC-3' SEQ ID NO. 36: 5'-GAATTCTTACTGGGATGCTCTTCG-3'
[0108]The PCR products were gel-purified and inserted into pCR2.1 TA cloning vector and sequence confirmed. The human Interferon-γ DNA sequence (SEQ ID NO. 21) and amino acid sequence (SEQ ID NO. 22) are shown in FIG. 1.
4. In Frame Fusion of HSA With Human IFN-α-1b, IFN-α-2b, IFN-β, IFN-ω or IFN-γ
[0109]Interferon analogs were made by fusion human albumin gene with interferon gene. There is a Bsu36 I site at the C'-terminus of HSA. All of the Interferons described in the Example section were fused into this site by PCR primer extension to generate a restriction enzyme site of Bsu36 I at the N-terminus of the Interferon DNA sequence. The Interferon DNA fragments were amplified by PCR and then subcloned into Bsu36 I and Xho I sites of pYZ-HSA or pYH-HSA vector which had been double digested with Bsu36 I and Xho I to linearize the plasmid DNA.
4.1. Construction of Vector Containing Interferon Analogs, HSA/INF-α-1b
[0110]Interferon-α-1b was fused to HAS C'-terminus by using the following PCR primers:
TABLE-US-00008 SEQ ID NO. 37: 5'-CTGCCTTAGGCTTATGTGATCTCCCTGAGACCC-3' and SEQ ID NO. 38: 5'-TCTCGAGTTACTTCCTCCTTAATCTTTC-3'.
(Human interferon-α-1b mature protein sequence is underlined in SEQ ID NO. 37).
[0111]A Xho I site (underlined in SEQ ID NO. 38) was created at the 3' end of interferon-α-1b gene. The PCR products were digested with Bsu361 and Xho I, and the fragment was gel purified and inserted into pYZ-HSA or pYH-HSA between of Bsu36 I and Xho I sites to generate a new plasmid DNA, pYZ-HSA/IFN-a. The HSA-hIFN-α-1b hybrid polynucleotide sequence (SEQ ID NO. 1) and its fusion protein amino acid sequence (SEQ ID NO. 2) are showed in FIG. 1.
4.2. Construction of Vector Containing Interferon Analogs, HSA/INF-α-2a and HSA/IFN-α-2b
[0112]Interferon-α-2a or Interferon-α-2b gene was fused to HSA C'-terminus by using the following PCR primers:
TABLE-US-00009 SEQ ID NO. 39: 5'-CTGCCTTAGGCTTATGTGATCTGCCTCAAACCC-3'.
[0113](Human Interferon-α-2a and Interferon-α-2b mature protein sequence is underlined), and
TABLE-US-00010 [0113]SEQ ID NO. 40: 5'-TCTCGAGTCATTCCTTACTTCTTAAAC-3'.
[0114]A Xho I site (underlined in SEQ ID NO. 40) was created at the 3' end of interferon-α gene. The PCR products were digested with Bsu36I and Xho I, and the fragment was gel purified and inserted into pYZ-HSA or pYH-HSA between of Bsu36 I and Xho I sites to generate a new plasmid DNA, pYZ-HSA/IFN-a. The HSA-hIFN-α-2b hybrid polynucleotide sequence (SEQ ID NO. 3) and its fusion protein amino acid sequence (SEQ ID NO. 4) are showed in FIG. 1.
4.3. Construction of Vector Containing Analog of Interferon-β, HSA/IFN-β
[0115]To make an analog of Interferon-1, HSA-IFN-β fusion protein, the following primers were designed SEQ ID NO. 41: 5'-CTGCCTTAGGCTTATACAACTTGCTTGGATTCC-3' (human interferon-β mature protein sequence underlined), and SEQ ID NO. 42: 5'-CACTCGAGTCAGTTTCGGAGGTAACC-3'
[0116](Xho I site underlined) and used to generate the modified human interferon-β DNA fragment. The PCR products were inserted between Bsu36I and Xho I sites of pYZ-HSA or pYH-HSA to generate a pYZ-HSA/IFN-β or pYH-HSA/IFN-β. The HSA-IFN-β hybrid polynucleotide sequence (SEQ ID NO. 5) and its fusion protein amino acid sequence (SEQ ID NO. 6) are shown in FIG. 1.
4.4. Construction of Vector Containing analog of Interferon-ω, HSA/IFN-ω
[0117]Human interferon-ω gene was fused with HSA DNA sequence by using two primers:
[0118]SEQ ID NO. 43: 5'-CTGCCTTAGGCTTATGTGATCTGCCTCAGAACCATGG-3' (Interferon-co mature protein sequence underlined), and
[0119]SEQ ID NO. 44: 5'-CTCGAGTCAAGATGAGCCCAGGTCTC-3'
[0120](Xho I site at the 3'-terminus of interferon-ω underlined).
[0121]The PCR products were gel purified and subcloned between Bsu36I and Xho I sites of pYZ-HAS or pYH-HSA to generate a pYZ-HSA/IFN-w or pYH-HSA/IFN-ω. The analog of interferon-ω, HSA-INF-ω hybrid polynucleotide, sequence (SEQ ID NO. 7) and its amino acid sequence (SEQ ID NO. 8) are shown in FIG. 1.
4.5. Construction of Vector Containing Analog of Interferon-γ, HSA/IFN-γ
[0122]The following primers:
[0123]SEQ ID NO. 45: 5'-ACTCCTTAGGCTTA CAGGACCCATATGTACAAGAAGC-3' (Interferon-γ mature protein sequence underlined), and SEQ ID NO. 46: 5'-CTCGAGTTACTGGGATGCTCTTCG-3' (Xho I site underlined) were used to modify Interferon-γ DNA sequence in order to subclone it into pYZ-HSA vector. PCR products were gel purified and double digested with Bsu36 I and Xho I and inserted between Bsu36 I and XhoI sites of pYZ-HSA, pYH-HSA to generate a pYZ-HSA/IFN-g, pYH-HSA/IFN-γ. The analog of Interferon-γ, HSA/IFN-γ hybrid polynucleotide, sequence (SEQ ID NO. 9) and its fusion protein amino acid sequence (SEQ ID NO. 10) are shown in FIG. 1.
5. Transformation of Yeast
[0124]An expression cassette contains, a promoter driving of a gene, here is the analog of Interferon, a terminator, and a selective marker (such as Zeocin, antibiotic selection; Histidine, a deficient selection). Yeast strains, GS115, SMD1168 or ZY101 are Histidine synthesis deficiency. When transform the Yeast with the linearized yeast transfer shuttle vector, the expression cassette will be inserted directly to the location with a homologue region recombination. Most time one cassette will be inserted into a yeast host. In here, we disclosed a novel method for making of a dual insertion of expression cassette into a different chromosome region by two vectors with two different select markers.
5.1. Single Expression Cassette Insertion on Yeast
[0125]A yeast Pichia pastoris strain, GS115, colony was inoculated into 5 ml of YPD medium in a 50 ml conical tube at 30° C. overnight with shaking at 250 rpm. 0.2 ml of the culture was inoculated into 500 ml of YPD medium continually shaking at 30° C. for further 2-3 hours or until the cell density reach to OD600=1.3-1.5. The cells were collected by centrifugation. The cell pellets were resuspend in 500 ml of ice-cold sterile water in order to wash the cells. After two rounds of washing, the cells were resuspended in 20 ml of ice-cold 1 M sorbitol to wash again and finally suspended in 1 ml of ice-cold IM sorbitol. The plasmid DNA constructs from Example 2, pYZ-HSA and in Example 4, pYZ-HSA/IFN-α-2a, pYZ-HSA/IFN-α-2b, pYZ-HSA/IFN-β, and pYZ-HSA/IFN-ω, pYZ-HSA/IFN-γ were linearized by PmeI restriction enzyme digestion first.
[0126]5 μg of each linear plasmid DNA was used to transform 80 μl of the freshly made yeast cells in an ice-cold 0.2 cm electroporation cuvette. The cells mixed with plasmid DNA were pulsed for 5-10 ms with field strength of 7500V/cm. After the pulse, 1 ml of ice-cold 1 M sorbitol was immediately added into the cuvette and the content was transferred to a sterile 15 ml tube. The transformed cells were incubated in 30° C. without shaking for 2 hours then spread on pre-made YPD-agar plates with 100 μg/ml Zeocin. The colonies were identified with the insert and the expression level by SDS-PAGE or western-blot with proper antibodies. Different strains of Pichia, such as X-33, KM71 and proteinase deficient strain SMD1168, ZY101 (Constructed and be used in manufacture of recombinant secretory protein drugs by yeast system, Zailin YU unpublished data 2002) were tested for the expression and secretory of recombinant proteins.
5.2. Dual Expression Cassette Insertion on Yeast
[0127]In order to gain a higher expression level, people are trying to select multi-insertion from the recombinant yeast (Invitrogen Corp), But the select is no efficient, we use a second transformation method on a yeast is carrying an expression cassette. To do this, for example, we use pYZ-HSA/IFN-β transformed yeast, the HSA/IFN-β expression cassette has inserted at AOX1 Gene location in yeast chromosome with a Zeocin resistance, transformed again with pYH-HSA/IFN-β expression cassette by the method described in section of 5.1 again. The new select marker will be on the YPD-Agar plate contains no Histidine (His.sup.-). Only the recombinant yeast contains the expression cassette with a Histidine gene can be survived in the medium. The new recombinant yeast now contains two genes of HSA/IFN-β, one located on AOX1 gene location, one is located on Histidinol dehydrogenase location. This recombinant yeast contains two selective markers and can grow in conditioned medium with antibiotic Zeocin, without the amino acid, Histidine, supplement.
[0128]By using this method, a different expression cassette also can be inserted to the yeast chromosome, such as the first expression cassette contains an interferon-a, and the second one is an interferon-γ; or the first expression cassette contains protein-X and the second expression cassette contains protein-X or protein different than first protein-X.
6. Secretion and Characterization of Interferon analogs Expressed by Pichia
[0129]Several colonies from each transformation of the Interferon analog, HSA-IFN, were cultured with Zeocin in the buffered minimal medium with glycerol overnight or until OD600=2-6 at 30° C. and shaking at 300 rpm. The cultured cells were collected by centrifuge at 1500 rpm for 5 minutes. Resuspend the cells into buffered minimal medium without glycerol and cell densities was keep in OD600=110 100% methanol was added into each flask to a final concentration at 0.5% every 24 hours to induce the protein expression. The culture medium was collected at different time points and the expression of each fusion protein was confirmed by SDS-PAGE and western blot. The results showed that human albumin and HSA-IFN fusion protein were expressed and secreted into the medium.
[0130]Mouse monoclonal anti-human serum albumin (Sigma) was used for immunoblotting on a SDS-PAGE gel. A typical Western blot experiment was carried on by electrophoresis transfer the protein from SDS-PAG to a nylon or nitrocellulose filter and incubated with a specific antibody (as the "first antibody"). Then an anti-first antibody would add to binding on the first antibody (as the "second antibody"). The second antibody was labeled with Fluorescence and the filter was exposed to an X-ray film. Protein molecular weight standard was used to determine the protein size. The results (FIG. 3) showed that the expressed recombinant proteins, HSA, Analog of Interferon-α (HSA-IFN-α-2a) therapeutic fusion protein, had an expected molecular weight and also had the same antigen as that of HSA prepared from a human blood plasma (Sigma). Using monoclonal anti-IFN-α specific antibody as first antibody, the HSA/Interferon-α fusion protein and human interferon-α (Chemicon International Inc. US) had the same antigen and showed that the molar ratio of HSA to interferon-α in the HAS/IFN-α-2a fusion protein is as expected (see Zailin YU Provisional Patent Application Ser. No. 60/392,948). Using monoclonal anti-Interferon-α specific antibody (CII, US) as first antibody, the HSA-IFN-α fusion protein and human Interferon-α (CII, US) had the same antigen and showed that the molar ratio of HSA to Interferon-β in the HSA/IFN-α fusion protein is as expected (FIG. 4).
7. Purification and Molecular Characterization of Interferon Analogs, HSA-IFNs
[0131]The cell culture medium (supernatant) containing the secreted protein of HSA or HSA-IFN fusion protein produced from the recombinant Pichia was collected, the salt concentration reduced, and the pH was adjusted to above 7.5. The concentrated sample was passed through an Affi-Gel Blue-gel (50-100 mesh) (Bio-Rad). The albumin or albumin fusion protein was bound to the matrix and eluded by a gradient 1-5M NaCl. 75-85% pure albumin or albumin-IFN can be recovered in this step. If further purification is necessary, a size exclusion chromatography is applied to give a 95-99% purity of proteins. The pyrogen was removed from the protein samples in order to meet the requirement for use in in vivo test. The Affi-Prep Polymyxin Support (BIO-Rad) column was used to remove endotoxin from the samples. The purified protein finally passed through 0.2 μM filter to be sterilized and the protein concentration was measured by a standard method by using a Bio-Rad Protein Assay Kit.
8. Viral Protection Assay of Interferon Analog, Human Interferon-α-2a
[0132]Antiviral activity of IFN-α-2a and its derivatives was determined by the capacity of the cytokine to protect human amnion WISH cells against vesicular stomatitis virus (VSV)-induced cytopathic effects (Rubinstein, et al., 1981, J. Virol. 37, 755-758). WISH cells (4.5×105 cells/ml) were seeded in a 96-well plate (100 μl/well) and incubated with 2-fold serial dilutions of IFN-α-2a or interferon analog, HSA/IFN-α-2a for 18 h at 37° C. WISH cell viability was determined by measuring the absorbance of crystal violet-stained cells in an ELISA plate. In this assay, native IFN-α-2a shows 50% protection of VSV-induced WISH cells (ED50) at a concentration of 0.45±0.04 pM. The IFN-α-2a analog exhibiting ED50 of 1.13±0.3 pM in this assay was considered as having 25% of the native antiviral potency (FIG. 5). Since HSA (65 kd) has a molecular weight about 3 times higher than that of interferon (19 kd), it can be inferred that HSA-IFN-α-2a fusion protein and Interferon analog have the same bioactivity as that of human Interferon-α-2a alone based on the molecular ratio.
9. Bioassay of Interferon-α analog, HSA/IFN-α, by ELISA
[0133]Enzyme-linked immunosorbent assay (ELISA) kit from Chemicon International, Inc. (California, US) was used for the quantitative determination of Interferon-concentration and bioactivities comparison with a commercial IFN-α sample. The IFN-α ELISA is based on the double-antibody sandwich method. With the ChemiKine® assay system, pre-coated goat anti-rabbit antibody plates are used to capture a specific IFN-α complex in each sample consisting of IFN-α antibody, biotinylated IFNα, and sample/standard. The biotinylated IFNα conjugate (competitive ligand), and sample or standard compete for IFNα specific antibody binding sites. Therefore, as the concentration of IFN-α in the sample increases, the amount of biotinylated IFNα captured by the antibody decreases. The assay is visualized using a streptavidin alkaline phosphatase conjugate and an ensuing chromagenic substrate reaction. The amount of IFNα detected in each sample is compared to an IFN-α standard curve which demonstrates an inverse relationship between Optical Density (O.D.) and cytokine concentration: i.e. the higher the O.D. the lower the cytokine concentration in the sample. The amount of color generated was directly proportional to the amount of conjugate bound to the IFN-α antibody complex, which, in turn, was directly proportional to the amount of IFN-α in the protein samples or standard. The absorbance of this complex was measured and a standard curve was generated by plotting absorbance versus the concentration of the IFN-α standards. The IFN-α concentration of the unknown sample was determined by comparing the optical density of the protein samples to the standard curve. The standards used in this assay were recombinant human IFN-α (with kit) calibrated against the Second International Reference Preparation (67/343), a urine-derived form of human IFN-α. Human recombinant IFN-α expressed in CHO cells was used as a control to determine the rHSA/IFN-α bio-activity.
[0134]The results showed that the bioactivity of IFN-α fused to HSA had same activity compared with the native Interferon-α. When in a higher concentration of HSA-IFN-α in a sample, the size of HSA-IFN-α fusion protein molecule may be too large, which prevents the anti-IFN-α antibody from efficiently binding to the IFN-α molecule fused to HSA, thereby the sensitivity of the detection in this bioassay would be reduced. Same results were observed in HSA-EPO ELISA experiments (YU and FU, US20040063635).
10. Stability Testing of Interferon Analogs, HSA-IFNs Fusion Proteins In Vitro
[0135]Using HSA/IFN-α-2a as an example, the stability of this interferon analog, HSA-interferons fusion protein, was tested at different time points at 37° C. and 50° C. 50 U (0.5 ng) of human interferon-α-2a from bacteria or 50 U (19.6 ng) of rHSA/IFN-α-2a was put into 200 μl thin-well PCR tube with 200 μl of tissue culture medium RPM1 without fetal bovine serum and other components. The tubes were sealed and left in water both. Samples were taken out at different time points and immediately put into -80° C. for storage. After all of samples were collected, a viral infection test on Wish cell line was carried out by standard protocols. The control of the test was set up in the same way as that in the bioassay. The results were showed that the "naked" human IFN-α lost almost all of its bioactivity after 10 hours at 37° C. (in FIG. 6 Panel A). But after 24 hours in 37° C., the bioactivity of Interferon Analog, HSA/IFN-α, still remained no changes. Experiment shows that even after 10 days, the antivirus potency has at least half remained. At 50° C. (Panel B), the "naked" human IFN-α lost its the bioactivity completely in 1 days. The Interferon Analog, HSA/IFN-α fusion protein, still retained near 90% of its bioactivity after 5 days. These results indicate that interferon analog may have a longer storage time and more resistant to degradation in harsh environment such as high temperatures.
11. Long Acting of Interferon Analogs in Plasma
[0136]Human Interferon Analogs, human serum albumin interferon-α-2b (HSA/IFN-α-2b) and Interferon-α-2b, were tested for the long acting bio-function or slow release in animal in vivo. 15 ng (about 1×103 U) human Interferon-α-2b plus 45 ng HSA (recombinant HAS from yeast) or 60 ng (about 1×103 U) human interferon-α-2b analog was injected into rats with 100 μl solution. After injection, the blood samples (0.05 ml) were collected. In the last day of experiments, a 05 ml of blood was collected from all the tested rats. The blood sample with EDTA added was spun in a microcentrafuge tube. Blood supernatant was collected and stored at -80° C. Using Chemicon International, Inc. (California, USA) Chemikine® Human IFNα EIA Kit (Cat# CYT102) all blood samples from the rats injected with interferon-α-2b (control) and Interferon Analog, HSA/IFN-α-2b were tested. The results showed that Interferon analog maintained much longer undigested status in plasma than the "naked" interferon-α-2b even with same amount of HSA injection (FIG. 7). The interferon-α-2b could only be detected from plasma in about 10 hours. The Interferon analog, HSA/IFN-α-2b could be detected even after 12 days of injection. This result is also consistent with the report that albumin has a half-life in plasma about 20 days (Waldmann T. A., in "Albumin Structure, Function and Uses", Rosenoer V. M. et al (eds), Pergamon Press, Oxford, 255-275, 1977). The instant novel form interferon analog shows a greater half-life in plasma. The plasma samples at day 12 were tested for their antiviral protection to WISH cells. The results showed that the control sample has no antiviral protection bio-function, but the instant interferon analog still maintains some bio-function in viral protection to the tested cells. This long acting bio-function gives interferon analogs novel utilities as a recombinant protein drugs for therapeutic treatment of patients.
12. Expression and Scale-Up of Interferon Analogs by Fermentation
[0137]In this example, it is shown that expression and scale-up are much easier by using a Pichia system than other currently available systems. After Pichia recombinants were isolated, expression of both Mut+ and Muts recombinants was tested. This involved growing a small culture of each recombinant, inducing with methanol, and taking sample at different time points. For secrete expression, both the cell pellet and supernatant were analyzed from each time point. The samples were analyzed on SDS-PAGE gels by using both Coomassie staining and Western blot. Bioactivities of expressed samples were tested and the expression levels and purity were monitored in each step for production of HSA fusion proteins.
REFERENCES
[0138]Brown, J. R. "Albumin structure, Function, and Uses" Pergamon, New York, 1977 [0139]Weikamp L, R, et al., Ann. Hum. Genet., 37 219-226, 1973 [0140]Carter D. C. et al., Science 244, 1195-1198, 1989 [0141]Waldmann T. A., in "Albumin Structure, Function and Uses", Rosenoer V. M. et al (eds), Pergamon Press, Oxford, 255-275, 1977 [0142]Shechter et al., Proc. Natl. Acad. Sci. USA. 2001 January 30; 98 (3): 1212-1217 [0143]O'Kelly, et al., 1985. Proc. Soc. Exp. Biol. Med. 178, 407-411 [0144]Rostaing, et al., 1998, J. Am. Soc. Nephrol. 9, 2344-2348 [0145]Vilcek (1991) "Interferons", in "Peptide Growth Factors and Their Receptors TI", edited by Spom and Roberts, Spring-Verlag Heidelberg, New York Inc., USA. pp 3-38
PATENT REFERENCES
EP 330 451
EP 361 991
[0146]U.S. Pat. No. 5,098,703U.S. Pat. No. 4,973,479U.S. Pat. No. 4,975,276U.S. Pat. No. 5,082,658U.S. Pat. No. 6,174,996U.S. Pat. No. 5,908,621U.S. Pat. No. 4,892,743U.S. Pat. No. 5,723,125U.S. Pat. No. 5,324,655U.S. Pat. No. 5,190,751
Sequence CWU
1
4612325DNAArtificial SequenceSynthesis 1atgaagtggg taacctttat ttcccttctt
tttctcttta gctcggctta ttccaggggt 60gtgtttcgtc gagatgcaca caagagtgag
gttgctcatc ggtttaaaga tttgggagaa 120gaaaatttca aagccttggt gttgattgcc
tttgctcagt atcttcagca gtgtccattt 180gaagatcatg taaaattagt gaatgaagta
actgaatttg caaaaacatg tgttgctgat 240gagtcagctg aaaattgtga caaatcactt
catacccttt ttggagacaa attatgcaca 300gttgcaactc ttcgtgaaac ctatggtgaa
atggctgact gctgtgcaaa acaagaacct 360gagagaaatg aatgcttctt gcaacacaaa
gatgacaacc caaacctccc ccgattggtg 420agaccagagg ttgatgtgat gtgcactgct
tttcatgaca atgaagagac atttttgaaa 480aaatacttat atgaaattgc cagaagacat
ccttactttt atgccccgga actccttttc 540tttgctaaaa ggtataaagc tgcttttaca
gaatgttgcc aagctgctga taaagctgcc 600tgcctgttgc caaagctcga tgaacttcgg
gatgaaggga aggcttcgtc tgccaaacag 660agactcaagt gtgccagtct ccaaaaattt
ggagaaagag ctttcaaagc atgggcagta 720gctcgcctga gccagagatt tcccaaagct
gagtttgcag aagtttccaa gttagtgaca 780gatcttacca aagtccacac ggaatgctgc
catggagatc tgcttgaatg tgctgatgac 840agggcggacc ttgccaagta tatctgtgaa
aatcaagatt cgatctccag taaactgaag 900gaatgctgtg aaaaacctct gttggaaaaa
tcccactgca ttgccgaagt ggaaaatgat 960gagatgcctg ctgacttgcc ttcattagct
gctgattttg ttgaaagtaa ggatgtttgc 1020aaaaactatg ctgaggcaaa ggatgtcttc
ctgggcatgt ttttgtatga atatgcaaga 1080aggcatcctg attactctgt cgtgctgctg
ctgagacttg ccaagacata tgaaaccact 1140ctagagaagt gctgtgccgc tgcagatcct
catgaatgct atgccaaagt gttcgatgaa 1200tttaaacctc ttgtggaaga gcctcagaat
ttaatcaaac aaaattgtga gctttttgag 1260cagcttggag agtacaaatt ccagaatgcg
ctattagttc gttacaccaa gaaagtaccc 1320caagtgtcaa ctccaactct tgtagaggtc
tcaagaaacc taggaaaagt gggcagcaaa 1380tgttgtaaac atcctgaagc aaaaagaatg
ccctgtgcag aagactatct atccgtggtc 1440ctgaaccagt tatgtgtgtt gcatgagaaa
acgccagtaa gtgacagagt caccaaatgc 1500tgcacagaat ccttggtgaa caggcgacca
tgcttttcag ctctggaagt cgatgaaaca 1560tacgttccca aagagtttaa tgctgaaaca
ttcaccttcc atgcagatat atgcacactt 1620tctgagaagg agagacaaat caagaaacaa
actgcacttg ttgagcttgt gaaacacaag 1680cccaaggcaa caaaagagca actgaaagct
gttatggatg atttcgcagc ttttgtagag 1740aagtgctgca aggctgacga taaggagacc
tgctttgccg aggagggtaa aaaacttgtt 1800gctgcaagtc aagctgcctt aggcttatgt
gatctccctg agacccacag cctggataac 1860aggaggacct tgatgctcct ggcacaaatg
agcagaatct ctccttcctc ctgtctgatg 1920gacagacatg actttggatt tccccaggag
gagtttgatg gcaaccagtt ccagaaggct 1980ccagccatct ctgtcctcca tgagctgatc
cagcagatct tcaacctctt taccacaaaa 2040gattcatctg ctgcttggga tgaggacctc
ctagacaaat tctgcaccga actctaccag 2100cagctgaatg acttggaagc ctgtgtgatg
caggaggaga gggtgggaga aactcccctg 2160atgaatgcgg actccatctt ggctgtgaag
aaatacttcc gaagaatcac tctctatctg 2220acagagaaga aatacagccc ttgtgcctgg
gaggttgtca gagcagaaat catgagatcc 2280ctctctttat caacaaactt gcaagaaaga
ttaaggagga agtaa 23252750PRTArtificial
SequenceSynthesis 2Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp
Leu Gly Glu1 5 10 15Glu
Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20
25 30Gln Cys Pro Phe Glu Asp His Val
Lys Leu Val Asn Glu Val Thr Glu 35 40
45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys
50 55 60Ser Leu His Thr Leu Phe Gly Asp
Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys
Gln Glu Pro 85 90 95Glu
Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu
100 105 110Pro Arg Leu Val Arg Pro Glu
Val Asp Val Met Cys Thr Ala Phe His 115 120
125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala
Arg 130 135 140Arg His Pro Tyr Phe Tyr
Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150
155 160Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala
Ala Asp Lys Ala Ala 165 170
175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser
180 185 190Ser Ala Lys Gln Arg Leu
Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg
Phe Pro 210 215 220Lys Ala Glu Phe Ala
Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys225 230
235 240Val His Thr Glu Cys Cys His Gly Asp Leu
Leu Glu Cys Ala Asp Asp 245 250
255Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser
260 265 270Ser Lys Leu Lys Glu
Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275
280 285Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala
Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305
310 315 320Glu Ala Lys Asp Val Phe Leu
Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325
330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg
Leu Ala Lys Thr 340 345 350Tyr
Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355
360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe
Lys Pro Leu Val Glu Glu Pro 370 375
380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385
390 395 400Tyr Lys Phe Gln
Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405
410 415Glu Val Ser Thr Pro Thr Leu Val Glu Val
Ser Arg Asn Leu Gly Lys 420 425
430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys
435 440 445Ala Glu Asp Tyr Leu Ser Val
Val Leu Asn Gln Leu Cys Val Leu His 450 455
460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu
Ser465 470 475 480Leu Val
Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr
485 490 495Tyr Val Pro Lys Glu Phe Asn
Ala Glu Thr Phe Thr Phe His Ala Asp 500 505
510Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln
Thr Ala 515 520 525Leu Val Glu Leu
Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530
535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu
Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575Ala Ala Ser Gln Ala
Ala Leu Gly Leu Cys Asp Leu Pro Glu Thr His 580
585 590Ser Leu Asp Asn Arg Arg Thr Leu Met Leu Leu Ala
Gln Met Ser Arg 595 600 605Ile Ser
Pro Ser Ser Cys Leu Met Asp Arg His Asp Phe Gly Phe Pro 610
615 620Gln Glu Glu Phe Asp Gly Asn Gln Phe Gln Lys
Ala Pro Ala Ile Ser625 630 635
640Val Leu His Glu Leu Ile Gln Gln Ile Phe Asn Leu Phe Thr Thr Lys
645 650 655Asp Ser Ser Ala
Ala Trp Asp Glu Asp Leu Leu Asp Lys Phe Cys Thr 660
665 670Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala
Cys Val Met Gln Glu 675 680 685Glu
Arg Val Gly Glu Thr Pro Leu Met Asn Ala Asp Ser Ile Leu Ala 690
695 700Val Lys Lys Tyr Phe Arg Arg Ile Thr Leu
Tyr Leu Thr Glu Lys Lys705 710 715
720Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg
Ser 725 730 735Leu Ser Leu
Ser Thr Asn Leu Gln Glu Arg Leu Arg Arg Lys 740
745 75032325DNAArtificial SequenceSynthess 3atgaagtggg
taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60gtgtttcgtc
gagatgcaca caagagtgag gttgctcatc ggtttaaaga tttgggagaa 120gaaaatttca
aagccttggt gttgattgcc tttgctcagt atcttcagca gtgtccattt 180gaagatcatg
taaaattagt gaatgaagta actgaatttg caaaaacatg tgttgctgat 240gagtcagctg
aaaattgtga caaatcactt catacccttt ttggagacaa attatgcaca 300gttgcaactc
ttcgtgaaac ctatggtgaa atggctgact gctgtgcaaa acaagaacct 360gagagaaatg
aatgcttctt gcaacacaaa gatgacaacc caaacctccc ccgattggtg 420agaccagagg
ttgatgtgat gtgcactgct tttcatgaca atgaagagac atttttgaaa 480aaatacttat
atgaaattgc cagaagacat ccttactttt atgccccgga actccttttc 540tttgctaaaa
ggtataaagc tgcttttaca gaatgttgcc aagctgctga taaagctgcc 600tgcctgttgc
caaagctcga tgaacttcgg gatgaaggga aggcttcgtc tgccaaacag 660agactcaagt
gtgccagtct ccaaaaattt ggagaaagag ctttcaaagc atgggcagta 720gctcgcctga
gccagagatt tcccaaagct gagtttgcag aagtttccaa gttagtgaca 780gatcttacca
aagtccacac ggaatgctgc catggagatc tgcttgaatg tgctgatgac 840agggcggacc
ttgccaagta tatctgtgaa aatcaagatt cgatctccag taaactgaag 900gaatgctgtg
aaaaacctct gttggaaaaa tcccactgca ttgccgaagt ggaaaatgat 960gagatgcctg
ctgacttgcc ttcattagct gctgattttg ttgaaagtaa ggatgtttgc 1020aaaaactatg
ctgaggcaaa ggatgtcttc ctgggcatgt ttttgtatga atatgcaaga 1080aggcatcctg
attactctgt cgtgctgctg ctgagacttg ccaagacata tgaaaccact 1140ctagagaagt
gctgtgccgc tgcagatcct catgaatgct atgccaaagt gttcgatgaa 1200tttaaacctc
ttgtggaaga gcctcagaat ttaatcaaac aaaattgtga gctttttgag 1260cagcttggag
agtacaaatt ccagaatgcg ctattagttc gttacaccaa gaaagtaccc 1320caagtgtcaa
ctccaactct tgtagaggtc tcaagaaacc taggaaaagt gggcagcaaa 1380tgttgtaaac
atcctgaagc aaaaagaatg ccctgtgcag aagactatct atccgtggtc 1440ctgaaccagt
tatgtgtgtt gcatgagaaa acgccagtaa gtgacagagt caccaaatgc 1500tgcacagaat
ccttggtgaa caggcgacca tgcttttcag ctctggaagt cgatgaaaca 1560tacgttccca
aagagtttaa tgctgaaaca ttcaccttcc atgcagatat atgcacactt 1620tctgagaagg
agagacaaat caagaaacaa actgcacttg ttgagcttgt gaaacacaag 1680cccaaggcaa
caaaagagca actgaaagct gttatggatg atttcgcagc ttttgtagag 1740aagtgctgca
aggctgacga taaggagacc tgctttgccg aggagggtaa aaaacttgtt 1800gctgcaagtc
aagctgcctt aggcttatgt gatctgcctc aaacccacag cctgggtagc 1860aggaggacct
tgatgctcct ggcacagatg aggaaaatct ctcttttctc ctgcttgaag 1920gacagacatg
actttggatt tccccaggag gagtttggca accagttcca aaaggctgaa 1980accatccctg
tcctccatga gatgatccag cagatcttca atctcttcag cacaaaggac 2040tcatctgctg
cttgggatga gaccctccta gacaaattct acactgaact ctaccagcag 2100ctgaatgacc
tggaagcctg tgtgatacag ggggtggggg tgacagagac tcccctgatg 2160aaggaggact
ccattctggc tgtgaggaaa tacttccaaa gaatcactct ctatctgaaa 2220gagaagaaat
acagcccttg tgcctgggag gttgtcagag cagaaatcat gagatctttt 2280tctttgtcaa
caaacttgca agaaagttta agaagtaagg aatga
23254750PRTArtificial SequenceSynthesis 4Asp Ala His Lys Ser Glu Val Ala
His Arg Phe Lys Asp Leu Gly Glu1 5 10
15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr
Leu Gln 20 25 30Gln Cys Pro
Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35
40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala
Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80Arg Glu Thr Tyr Gly Glu Met
Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90
95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn
Pro Asn Leu 100 105 110Pro Arg
Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115
120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr
Leu Tyr Glu Ile Ala Arg 130 135 140Arg
His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145
150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165
170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu
Gly Lys Ala Ser 180 185 190Ser
Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195
200 205Arg Ala Phe Lys Ala Trp Ala Val Ala
Arg Leu Ser Gln Arg Phe Pro 210 215
220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys225
230 235 240Val His Thr Glu
Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245
250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu
Asn Gln Asp Ser Ile Ser 260 265
270Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His
275 280 285Cys Ile Ala Glu Val Glu Asn
Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295
300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr
Ala305 310 315 320Glu Ala
Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val
Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345
350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro
His Glu 355 360 365Cys Tyr Ala Lys
Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370
375 380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu
Gln Leu Gly Glu385 390 395
400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro
405 410 415Glu Val Ser Thr Pro
Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420
425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys
Arg Met Pro Cys 435 440 445Ala Glu
Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450
455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys
Cys Cys Thr Glu Ser465 470 475
480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr
485 490 495Tyr Val Pro Lys
Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500
505 510Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile
Lys Lys Gln Thr Ala 515 520 525Leu
Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530
535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe
Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu
Val 565 570 575Ala Ala Ser
Gln Ala Ala Leu Gly Leu Cys Asp Leu Pro Gln Thr His 580
585 590Ser Leu Gly Ser Arg Arg Thr Leu Met Leu
Leu Ala Gln Met Arg Lys 595 600
605Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro 610
615 620Gln Glu Glu Phe Gly Asn Gln Phe
Gln Lys Ala Glu Thr Ile Pro Val625 630
635 640Leu His Glu Met Ile Gln Gln Ile Phe Asn Leu Phe
Ser Thr Lys Asp 645 650
655Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu
660 665 670Leu Tyr Gln Gln Leu Asn
Asp Leu Glu Ala Cys Val Ile Gln Gly Val 675 680
685Gly Val Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu
Ala Val 690 695 700Arg Lys Tyr Phe Gln
Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr705 710
715 720Ser Pro Cys Ala Trp Glu Val Val Arg Ala
Glu Ile Met Arg Ser Phe 725 730
735Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu Arg Ser Lys Glu
740 745 75052322DNAArtificial
SequenceSynthesis 5atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta
ttccaggggt 60gtgtttcgtc gagatgcaca caagagtgag gttgctcatc ggtttaaaga
tttgggagaa 120gaaaatttca aagccttggt gttgattgcc tttgctcagt atcttcagca
gtgtccattt 180gaagatcatg taaaattagt gaatgaagta actgaatttg caaaaacatg
tgttgctgat 240gagtcagctg aaaattgtga caaatcactt catacccttt ttggagacaa
attatgcaca 300gttgcaactc ttcgtgaaac ctatggtgaa atggctgact gctgtgcaaa
acaagaacct 360gagagaaatg aatgcttctt gcaacacaaa gatgacaacc caaacctccc
ccgattggtg 420agaccagagg ttgatgtgat gtgcactgct tttcatgaca atgaagagac
atttttgaaa 480aaatacttat atgaaattgc cagaagacat ccttactttt atgccccgga
actccttttc 540tttgctaaaa ggtataaagc tgcttttaca gaatgttgcc aagctgctga
taaagctgcc 600tgcctgttgc caaagctcga tgaacttcgg gatgaaggga aggcttcgtc
tgccaaacag 660agactcaagt gtgccagtct ccaaaaattt ggagaaagag ctttcaaagc
atgggcagta 720gctcgcctga gccagagatt tcccaaagct gagtttgcag aagtttccaa
gttagtgaca 780gatcttacca aagtccacac ggaatgctgc catggagatc tgcttgaatg
tgctgatgac 840agggcggacc ttgccaagta tatctgtgaa aatcaagatt cgatctccag
taaactgaag 900gaatgctgtg aaaaacctct gttggaaaaa tcccactgca ttgccgaagt
ggaaaatgat 960gagatgcctg ctgacttgcc ttcattagct gctgattttg ttgaaagtaa
ggatgtttgc 1020aaaaactatg ctgaggcaaa ggatgtcttc ctgggcatgt ttttgtatga
atatgcaaga 1080aggcatcctg attactctgt cgtgctgctg ctgagacttg ccaagacata
tgaaaccact 1140ctagagaagt gctgtgccgc tgcagatcct catgaatgct atgccaaagt
gttcgatgaa 1200tttaaacctc ttgtggaaga gcctcagaat ttaatcaaac aaaattgtga
gctttttgag 1260cagcttggag agtacaaatt ccagaatgcg ctattagttc gttacaccaa
gaaagtaccc 1320caagtgtcaa ctccaactct tgtagaggtc tcaagaaacc taggaaaagt
gggcagcaaa 1380tgttgtaaac atcctgaagc aaaaagaatg ccctgtgcag aagactatct
atccgtggtc 1440ctgaaccagt tatgtgtgtt gcatgagaaa acgccagtaa gtgacagagt
caccaaatgc 1500tgcacagaat ccttggtgaa caggcgacca tgcttttcag ctctggaagt
cgatgaaaca 1560tacgttccca aagagtttaa tgctgaaaca ttcaccttcc atgcagatat
atgcacactt 1620tctgagaagg agagacaaat caagaaacaa actgcacttg ttgagcttgt
gaaacacaag 1680cccaaggcaa caaaagagca actgaaagct gttatggatg atttcgcagc
ttttgtagag 1740aagtgctgca aggctgacga taaggagacc tgctttgccg aggagggtaa
aaaacttgtt 1800gctgcaagtc aagctgcctt aggcttatac aacttgcttg gattcctaca
aagaagcagc 1860aattttcagt gtcagaagct cctgtggcaa ttgaatggga ggcttgaata
ctgcctcaag 1920gacaggatga actttgacat ccctgaggag attaagcagc tgcagcagtt
ccagaaggag 1980gacgccgcat tgaccatcta tgagatgctc cagaacatct ttgctatttt
cagacaagat 2040tcatctagca ctggctggaa tgagactatt gttgagaacc tcctggctaa
tgtctatcat 2100cagataaacc atctgaagac agtcctggaa gaaaaactgg agaaagaaga
tttcaccagg 2160ggaaaactca tgagcagtct gcacctgaaa agatattatg ggaggattct
gcattacctg 2220aaggccaagg agtacagtca ctgtgcctgg accatagtca gagtggaaat
cctaaggaac 2280ttttacttca ttaacagact tacaggttac ctccgaaact ga
23226749PRTArtificial SequenceSynthesis 6Asp Ala His Lys Ser
Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5
10 15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe
Ala Gln Tyr Leu Gln 20 25
30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu
35 40 45Phe Ala Lys Thr Cys Val Ala Asp
Glu Ser Ala Glu Asn Cys Asp Lys 50 55
60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80Arg Glu Thr Tyr Gly
Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85
90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp
Asp Asn Pro Asn Leu 100 105
110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His
115 120 125Asp Asn Glu Glu Thr Phe Leu
Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135
140Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys
Arg145 150 155 160Tyr Lys
Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
165 170 175Cys Leu Leu Pro Lys Leu Asp
Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185
190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe
Gly Glu 195 200 205Arg Ala Phe Lys
Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210
215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr
Asp Leu Thr Lys225 230 235
240Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp
245 250 255Arg Ala Asp Leu Ala
Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260
265 270Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu
Glu Lys Ser His 275 280 285Cys Ile
Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290
295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val
Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp
Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340
345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala
Ala Asp Pro His Glu 355 360 365Cys
Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370
375 380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu
Phe Glu Gln Leu Gly Glu385 390 395
400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val
Pro 405 410 415Glu Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420
425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu
Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450
455 460Glu Lys Thr Pro Val Ser Asp Arg
Val Thr Lys Cys Cys Thr Glu Ser465 470
475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu
Val Asp Glu Thr 485 490
495Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp
500 505 510Ile Cys Thr Leu Ser Glu
Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520
525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu
Gln Leu 530 535 540Lys Ala Val Met Asp
Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550
555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu
Glu Gly Lys Lys Leu Val 565 570
575Ala Ala Ser Gln Ala Ala Leu Gly Leu Tyr Asn Leu Leu Gly Phe Leu
580 585 590Gln Arg Ser Ser Asn
Phe Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn 595
600 605Gly Arg Leu Glu Tyr Cys Leu Lys Asp Arg Met Asn
Phe Asp Ile Pro 610 615 620Glu Glu Ile
Lys Gln Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu625
630 635 640Thr Ile Tyr Glu Met Leu Gln
Asn Ile Phe Ala Ile Phe Arg Gln Asp 645
650 655Ser Ser Ser Thr Gly Trp Asn Glu Thr Ile Val Glu
Asn Leu Leu Ala 660 665 670Asn
Val Tyr His Gln Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys 675
680 685Leu Glu Lys Glu Asp Phe Thr Arg Gly
Lys Leu Met Ser Ser Leu His 690 695
700Leu Lys Arg Tyr Tyr Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu705
710 715 720Tyr Ser His Cys
Ala Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn 725
730 735Phe Tyr Phe Ile Asn Arg Leu Thr Gly Tyr
Leu Arg Asn 740 74572346DNAArtificial
SequenceSynthesis 7atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta
ttccaggggt 60gtgtttcgtc gagatgcaca caagagtgag gttgctcatc ggtttaaaga
tttgggagaa 120gaaaatttca aagccttggt gttgattgcc tttgctcagt atcttcagca
gtgtccattt 180gaagatcatg taaaattagt gaatgaagta actgaatttg caaaaacatg
tgttgctgat 240gagtcagctg aaaattgtga caaatcactt catacccttt ttggagacaa
attatgcaca 300gttgcaactc ttcgtgaaac ctatggtgaa atggctgact gctgtgcaaa
acaagaacct 360gagagaaatg aatgcttctt gcaacacaaa gatgacaacc caaacctccc
ccgattggtg 420agaccagagg ttgatgtgat gtgcactgct tttcatgaca atgaagagac
atttttgaaa 480aaatacttat atgaaattgc cagaagacat ccttactttt atgccccgga
actccttttc 540tttgctaaaa ggtataaagc tgcttttaca gaatgttgcc aagctgctga
taaagctgcc 600tgcctgttgc caaagctcga tgaacttcgg gatgaaggga aggcttcgtc
tgccaaacag 660agactcaagt gtgccagtct ccaaaaattt ggagaaagag ctttcaaagc
atgggcagta 720gctcgcctga gccagagatt tcccaaagct gagtttgcag aagtttccaa
gttagtgaca 780gatcttacca aagtccacac ggaatgctgc catggagatc tgcttgaatg
tgctgatgac 840agggcggacc ttgccaagta tatctgtgaa aatcaagatt cgatctccag
taaactgaag 900gaatgctgtg aaaaacctct gttggaaaaa tcccactgca ttgccgaagt
ggaaaatgat 960gagatgcctg ctgacttgcc ttcattagct gctgattttg ttgaaagtaa
ggatgtttgc 1020aaaaactatg ctgaggcaaa ggatgtcttc ctgggcatgt ttttgtatga
atatgcaaga 1080aggcatcctg attactctgt cgtgctgctg ctgagacttg ccaagacata
tgaaaccact 1140ctagagaagt gctgtgccgc tgcagatcct catgaatgct atgccaaagt
gttcgatgaa 1200tttaaacctc ttgtggaaga gcctcagaat ttaatcaaac aaaattgtga
gctttttgag 1260cagcttggag agtacaaatt ccagaatgcg ctattagttc gttacaccaa
gaaagtaccc 1320caagtgtcaa ctccaactct tgtagaggtc tcaagaaacc taggaaaagt
gggcagcaaa 1380tgttgtaaac atcctgaagc aaaaagaatg ccctgtgcag aagactatct
atccgtggtc 1440ctgaaccagt tatgtgtgtt gcatgagaaa acgccagtaa gtgacagagt
caccaaatgc 1500tgcacagaat ccttggtgaa caggcgacca tgcttttcag ctctggaagt
cgatgaaaca 1560tacgttccca aagagtttaa tgctgaaaca ttcaccttcc atgcagatat
atgcacactt 1620tctgagaagg agagacaaat caagaaacaa actgcacttg ttgagcttgt
gaaacacaag 1680cccaaggcaa caaaagagca actgaaagct gttatggatg atttcgcagc
ttttgtagag 1740aagtgctgca aggctgacga taaggagacc tgctttgccg aggagggtaa
aaaacttgtt 1800gctgcaagtc aagctgcctt aggcttatgt gatctgcctc agaaccatgg
cctacttagc 1860aggaacacct tggtgcttct gcaccaaatg aggagaatct cccctttctt
gtgtctcaag 1920gacagaagag acttcaggtt cccccaggag atggtaaaag ggagccagtt
gcagaaggcc 1980catgtcatgt ctgtcctcca tgagatgctg cagcagatct tcagcctctt
ccacacagag 2040cgctcctctg ctgcctggaa catgaccctc ctagaccaac tccacactgg
acttcatcag 2100caactgcaac acctggagac ctgcttgctg caggtagtgg gagaaggaga
atctgctggg 2160gcaattagca gccctgcact gaccttgagg aggtacttcc agggaatccg
tgtctacctg 2220aaagagaaga aatacagcga ctgtgcctgg gaagttgtca gaatggaaat
catgaaatcc 2280ttgttcttat caacaaacat gcaagaaaga ctgagaagta aagatagaga
cctgggctca 2340tcttga
23468757PRTArtificial Sequencesynthesis 8Asp Ala His Lys Ser
Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5
10 15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe
Ala Gln Tyr Leu Gln 20 25
30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu
35 40 45Phe Ala Lys Thr Cys Val Ala Asp
Glu Ser Ala Glu Asn Cys Asp Lys 50 55
60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80Arg Glu Thr Tyr Gly
Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85
90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp
Asp Asn Pro Asn Leu 100 105
110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His
115 120 125Asp Asn Glu Glu Thr Phe Leu
Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135
140Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys
Arg145 150 155 160Tyr Lys
Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
165 170 175Cys Leu Leu Pro Lys Leu Asp
Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185
190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe
Gly Glu 195 200 205Arg Ala Phe Lys
Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210
215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr
Asp Leu Thr Lys225 230 235
240Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp
245 250 255Arg Ala Asp Leu Ala
Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260
265 270Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu
Glu Lys Ser His 275 280 285Cys Ile
Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290
295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val
Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp
Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340
345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala
Ala Asp Pro His Glu 355 360 365Cys
Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370
375 380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu
Phe Glu Gln Leu Gly Glu385 390 395
400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val
Pro 405 410 415Glu Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420
425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu
Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450
455 460Glu Lys Thr Pro Val Ser Asp Arg
Val Thr Lys Cys Cys Thr Glu Ser465 470
475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu
Val Asp Glu Thr 485 490
495Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp
500 505 510Ile Cys Thr Leu Ser Glu
Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520
525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu
Gln Leu 530 535 540Lys Ala Val Met Asp
Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550
555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu
Glu Gly Lys Lys Leu Val 565 570
575Ala Ala Ser Gln Ala Ala Leu Gly Leu Cys Asp Leu Pro Gln Asn His
580 585 590Gly Leu Leu Ser Arg
Asn Thr Leu Val Leu Leu His Gln Met Arg Arg 595
600 605Ile Ser Pro Phe Leu Cys Leu Lys Asp Arg Arg Asp
Phe Arg Phe Pro 610 615 620Gln Glu Met
Val Lys Gly Ser Gln Leu Gln Lys Ala His Val Met Ser625
630 635 640Val Leu His Glu Met Leu Gln
Gln Ile Phe Ser Leu Phe His Thr Glu 645
650 655Arg Ser Ser Ala Ala Trp Asn Met Thr Leu Leu Asp
Gln Leu His Thr 660 665 670Gly
Leu His Gln Gln Leu Gln His Leu Glu Thr Cys Leu Leu Gln Val 675
680 685Val Gly Glu Gly Glu Ser Ala Gly Ala
Ile Ser Ser Pro Ala Leu Thr 690 695
700Leu Arg Arg Tyr Phe Gln Gly Ile Arg Val Tyr Leu Lys Glu Lys Lys705
710 715 720Tyr Ser Asp Cys
Ala Trp Glu Val Val Arg Met Glu Ile Met Lys Ser 725
730 735Leu Phe Leu Ser Thr Asn Met Gln Glu Arg
Leu Arg Ser Lys Asp Arg 740 745
750Asp Leu Gly Ser Ser 75592259DNAArtificial Sequencesynthesis
9atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt
60gtgtttcgtc gagatgcaca caagagtgag gttgctcatc ggtttaaaga tttgggagaa
120gaaaatttca aagccttggt gttgattgcc tttgctcagt atcttcagca gtgtccattt
180gaagatcatg taaaattagt gaatgaagta actgaatttg caaaaacatg tgttgctgat
240gagtcagctg aaaattgtga caaatcactt catacccttt ttggagacaa attatgcaca
300gttgcaactc ttcgtgaaac ctatggtgaa atggctgact gctgtgcaaa acaagaacct
360gagagaaatg aatgcttctt gcaacacaaa gatgacaacc caaacctccc ccgattggtg
420agaccagagg ttgatgtgat gtgcactgct tttcatgaca atgaagagac atttttgaaa
480aaatacttat atgaaattgc cagaagacat ccttactttt atgccccgga actccttttc
540tttgctaaaa ggtataaagc tgcttttaca gaatgttgcc aagctgctga taaagctgcc
600tgcctgttgc caaagctcga tgaacttcgg gatgaaggga aggcttcgtc tgccaaacag
660agactcaagt gtgccagtct ccaaaaattt ggagaaagag ctttcaaagc atgggcagta
720gctcgcctga gccagagatt tcccaaagct gagtttgcag aagtttccaa gttagtgaca
780gatcttacca aagtccacac ggaatgctgc catggagatc tgcttgaatg tgctgatgac
840agggcggacc ttgccaagta tatctgtgaa aatcaagatt cgatctccag taaactgaag
900gaatgctgtg aaaaacctct gttggaaaaa tcccactgca ttgccgaagt ggaaaatgat
960gagatgcctg ctgacttgcc ttcattagct gctgattttg ttgaaagtaa ggatgtttgc
1020aaaaactatg ctgaggcaaa ggatgtcttc ctgggcatgt ttttgtatga atatgcaaga
1080aggcatcctg attactctgt cgtgctgctg ctgagacttg ccaagacata tgaaaccact
1140ctagagaagt gctgtgccgc tgcagatcct catgaatgct atgccaaagt gttcgatgaa
1200tttaaacctc ttgtggaaga gcctcagaat ttaatcaaac aaaattgtga gctttttgag
1260cagcttggag agtacaaatt ccagaatgcg ctattagttc gttacaccaa gaaagtaccc
1320caagtgtcaa ctccaactct tgtagaggtc tcaagaaacc taggaaaagt gggcagcaaa
1380tgttgtaaac atcctgaagc aaaaagaatg ccctgtgcag aagactatct atccgtggtc
1440ctgaaccagt tatgtgtgtt gcatgagaaa acgccagtaa gtgacagagt caccaaatgc
1500tgcacagaat ccttggtgaa caggcgacca tgcttttcag ctctggaagt cgatgaaaca
1560tacgttccca aagagtttaa tgctgaaaca ttcaccttcc atgcagatat atgcacactt
1620tctgagaagg agagacaaat caagaaacaa actgcacttg ttgagcttgt gaaacacaag
1680cccaaggcaa caaaagagca actgaaagct gttatggatg atttcgcagc ttttgtagag
1740aagtgctgca aggctgacga taaggagacc tgctttgccg aggagggtaa aaaacttgtt
1800gctgcaagtc aagctgcctt aggcttacag gacccatatg tacaagaagc agaaaacctt
1860aagaaatatt ttaatgcagg tcattcagat gtagcggata atggaactct tttcttaggc
1920attttgaaga attggaaaga ggagagtgac agaaaaataa tgcagagcca aattgtctcc
1980ttttacttca aactttttaa aaactttaaa gatgaccaga gcatccaaaa gagtgtggag
2040accatcaagg aagacatgaa tgtcaagttt ttcaatagca acaaaaagaa acgagatgac
2100ttcgaaaagc tgactaatta ttcggtaact gacttgaatg tccaacgcaa agcaatacat
2160gaactcatcc aagtgatggc tgaactgtcg ccagcagcta aaacagggaa gcgaaaaagg
2220agtcagatgc tgtttcgagg tcgaagagca tcccagtaa
225910728PRTArtificial Sequencesynthesis 10Asp Ala His Lys Ser Glu Val
Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10
15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln
Tyr Leu Gln 20 25 30Gln Cys
Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35
40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser
Ala Glu Asn Cys Asp Lys 50 55 60Ser
Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80Arg Glu Thr Tyr Gly Glu
Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85
90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp
Asn Pro Asn Leu 100 105 110Pro
Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115
120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys
Tyr Leu Tyr Glu Ile Ala Arg 130 135
140Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145
150 155 160Tyr Lys Ala Ala
Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165
170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg
Asp Glu Gly Lys Ala Ser 180 185
190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu
195 200 205Arg Ala Phe Lys Ala Trp Ala
Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215
220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr
Lys225 230 235 240Val His
Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp
245 250 255Arg Ala Asp Leu Ala Lys Tyr
Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265
270Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys
Ser His 275 280 285Cys Ile Ala Glu
Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290
295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys
Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr
Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340
345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr
Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370
375 380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe
Glu Gln Leu Gly Glu385 390 395
400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro
405 410 415Glu Val Ser Thr
Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420
425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala
Lys Arg Met Pro Cys 435 440 445Ala
Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450
455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr
Lys Cys Cys Thr Glu Ser465 470 475
480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu
Thr 485 490 495Tyr Val Pro
Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500
505 510Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln
Ile Lys Lys Gln Thr Ala 515 520
525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530
535 540Lys Ala Val Met Asp Asp Phe Ala
Ala Phe Val Glu Lys Cys Cys Lys545 550
555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly
Lys Lys Leu Val 565 570
575Ala Ala Ser Gln Ala Ala Leu Gly Leu Gln Asp Pro Tyr Val Gln Glu
580 585 590Ala Glu Asn Leu Lys Lys
Tyr Phe Asn Ala Gly His Ser Asp Val Ala 595 600
605Asp Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys
Glu Glu 610 615 620Ser Asp Arg Lys Ile
Met Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys625 630
635 640Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser
Ile Gln Lys Ser Val Glu 645 650
655Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys
660 665 670Lys Arg Asp Asp Phe
Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu 675
680 685Asn Val Gln Arg Lys Ala Ile His Glu Leu Ile Gln
Val Met Ala Glu 690 695 700Leu Ser Pro
Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gln Met Leu705
710 715 720Phe Arg Gly Arg Arg Ala Ser
Gln 725111830DNAHomo sapiens 11atgaagtggg taacctttat
ttcccttctt tttctcttta gctcggctta ttccaggggt 60gtgtttcgtc gagatgcaca
caagagtgag gttgctcatc ggtttaaaga tttgggagaa 120gaaaatttca aagccttggt
gttgattgcc tttgctcagt atcttcagca gtgtccattt 180gaagatcatg taaaattagt
gaatgaagta actgaatttg caaaaacatg tgttgctgat 240gagtcagctg aaaattgtga
caaatcactt catacccttt ttggagacaa attatgcaca 300gttgcaactc ttcgtgaaac
ctatggtgaa atggctgact gctgtgcaaa acaagaacct 360gagagaaatg aatgcttctt
gcaacacaaa gatgacaacc caaacctccc ccgattggtg 420agaccagagg ttgatgtgat
gtgcactgct tttcatgaca atgaagagac atttttgaaa 480aaatacttat atgaaattgc
cagaagacat ccttactttt atgccccgga actccttttc 540tttgctaaaa ggtataaagc
tgcttttaca gaatgttgcc aagctgctga taaagctgcc 600tgcctgttgc caaagctcga
tgaacttcgg gatgaaggga aggcttcgtc tgccaaacag 660agactcaagt gtgccagtct
ccaaaaattt ggagaaagag ctttcaaagc atgggcagta 720gctcgcctga gccagagatt
tcccaaagct gagtttgcag aagtttccaa gttagtgaca 780gatcttacca aagtccacac
ggaatgctgc catggagatc tgcttgaatg tgctgatgac 840agggcggacc ttgccaagta
tatctgtgaa aatcaagatt cgatctccag taaactgaag 900gaatgctgtg aaaaacctct
gttggaaaaa tcccactgca ttgccgaagt ggaaaatgat 960gagatgcctg ctgacttgcc
ttcattagct gctgattttg ttgaaagtaa ggatgtttgc 1020aaaaactatg ctgaggcaaa
ggatgtcttc ctgggcatgt ttttgtatga atatgcaaga 1080aggcatcctg attactctgt
cgtgctgctg ctgagacttg ccaagacata tgaaaccact 1140ctagagaagt gctgtgccgc
tgcagatcct catgaatgct atgccaaagt gttcgatgaa 1200tttaaacctc ttgtggaaga
gcctcagaat ttaatcaaac aaaattgtga gctttttgag 1260cagcttggag agtacaaatt
ccagaatgcg ctattagttc gttacaccaa gaaagtaccc 1320caagtgtcaa ctccaactct
tgtagaggtc tcaagaaacc taggaaaagt gggcagcaaa 1380tgttgtaaac atcctgaagc
aaaaagaatg ccctgtgcag aagactatct atccgtggtc 1440ctgaaccagt tatgtgtgtt
gcatgagaaa acgccagtaa gtgacagagt caccaaatgc 1500tgcacagaat ccttggtgaa
caggcgacca tgcttttcag ctctggaagt cgatgaaaca 1560tacgttccca aagagtttaa
tgctgaaaca ttcaccttcc atgcagatat atgcacactt 1620tctgagaagg agagacaaat
caagaaacaa actgcacttg ttgagcttgt gaaacacaag 1680cccaaggcaa caaaagagca
actgaaagct gttatggatg atttcgcagc ttttgtagag 1740aagtgctgca aggctgacga
taaggagacc tgctttgccg aggagggtaa aaaacttgtt 1800gctgcaagtc aagctgcctt
aggcttataa 183012585PRTHomo sapiens
12Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1
5 10 15Glu Asn Phe Lys Ala Leu
Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25
30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu
Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50
55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr
Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe
Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100
105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys
Thr Ala Phe His 115 120 125Asp Asn
Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130
135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu
Phe Phe Ala Lys Arg145 150 155
160Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180
185 190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu
Gln Lys Phe Gly Glu 195 200 205Arg
Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210
215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu
Val Thr Asp Leu Thr Lys225 230 235
240Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp
Asp 245 250 255Arg Ala Asp
Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260
265 270Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro
Leu Leu Glu Lys Ser His 275 280
285Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290
295 300Leu Ala Ala Asp Phe Val Glu Ser
Lys Asp Val Cys Lys Asn Tyr Ala305 310
315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr
Glu Tyr Ala Arg 325 330
335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr
340 345 350Tyr Glu Thr Thr Leu Glu
Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360
365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu
Glu Pro 370 375 380Gln Asn Leu Ile Lys
Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390
395 400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg
Tyr Thr Lys Lys Val Pro 405 410
415Glu Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys
420 425 430Val Gly Ser Lys Cys
Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435
440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu
Cys Val Leu His 450 455 460Glu Lys Thr
Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser465
470 475 480Leu Val Asn Arg Arg Pro Cys
Phe Ser Ala Leu Glu Val Asp Glu Thr 485
490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr
Phe His Ala Asp 500 505 510Ile
Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515
520 525Leu Val Glu Leu Val Lys His Lys Pro
Lys Ala Thr Lys Glu Gln Leu 530 535
540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545
550 555 560Ala Asp Asp Lys
Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565
570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu
580 58513567DNAHomo sapiens 13atggccttga cctttgcttt
actggtggcc ctcctggtgc tcagctgcaa gtcaagctgc 60tctgtgggct gtgatctgcc
tcaaacccac agcctgggta gcaggaggac cttgatgctc 120ctggcacaga tgaggagaat
ctctcttttc tcctgcttga aggacagaca tgactttgga 180tttccccagg aggagtttgg
caaccagttc caaaaggctg aaaccatccc tgtcctccat 240gagatgatcc agcagatctt
caatctcttc agcacaaagg actcatctgc tgcttgggat 300gagaccctcc tagacaaatt
ctacactgaa ctctaccagc agctgaatga cctggaagcc 360tgtgtgatac agggggtggg
ggtgacagag actcccctga tgaaggagga ctccattctg 420gctgtgagga aatacttcca
aagaatcact ctctatctga aagagaagaa atacagccct 480tgtgcctggg aggttgtcag
agcagaaatc atgagatctt tttctttgtc aacaaacttg 540caagaaagtt taagaagtaa
ggaatga 56714188PRTHomo sapiens
14Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys1
5 10 15Lys Ser Ser Cys Ser Val
Gly Cys Asp Leu Pro Gln Thr His Ser Leu 20 25
30Gly Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg
Arg Ile Ser 35 40 45Leu Phe Ser
Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu 50
55 60Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile
Pro Val Leu His65 70 75
80Glu Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser
85 90 95Ala Ala Trp Asp Glu Thr
Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr 100
105 110Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln
Gly Val Gly Val 115 120 125Thr Glu
Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys 130
135 140Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu
Lys Lys Tyr Ser Pro145 150 155
160Cys Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu
165 170 175Ser Thr Asn Leu
Gln Glu Ser Leu Arg Ser Lys Glu 180
18515567DNAHomo sapiens 15atggccttga cctttgcttt actggtggcc ctcctggtgc
tcagctgcaa gtcaagctgc 60tctgtgggct gtgatctgcc tcaaacccac agcctgggta
gcaggaggac cttgatgctc 120ctggcacaga tgaggaaaat ctctcttttc tcctgcttga
aggacagaca tgactttgga 180tttccccagg aggagtttgg caaccagttc caaaaggctg
aaaccatccc tgtcctccat 240gagatgatcc agcagatctt caatctcttc agcacaaagg
actcatctgc tgcttgggat 300gagaccctcc tagacaaatt ctacactgaa ctctaccagc
agctgaatga cctggaagcc 360tgtgtgatac agggggtggg ggtgacagag actcccctga
tgaaggagga ctccattctg 420gctgtgagga aatacttcca aagaatcact ctctatctga
aagagaagaa atacagccct 480tgtgcctggg aggttgtcag agcagaaatc atgagatctt
tttctttgtc aacaaacttg 540caagaaagtt taagaagtaa ggaatga
56716188PRTHomo sapiens 16Met Ala Leu Thr Phe Ala
Leu Leu Val Ala Leu Leu Val Leu Ser Cys1 5
10 15Lys Ser Ser Cys Ser Val Gly Cys Asp Leu Pro Gln
Thr His Ser Leu 20 25 30Gly
Ser Arg Arg Thr Leu Met Leu Leu Ala Gln Met Arg Lys Ile Ser 35
40 45Leu Phe Ser Cys Leu Lys Asp Arg His
Asp Phe Gly Phe Pro Gln Glu 50 55
60Glu Phe Gly Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His65
70 75 80Glu Met Ile Gln Gln
Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser 85
90 95Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe
Tyr Thr Glu Leu Tyr 100 105
110Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val
115 120 125Thr Glu Thr Pro Leu Met Lys
Glu Asp Ser Ile Leu Ala Val Arg Lys 130 135
140Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser
Pro145 150 155 160Cys Ala
Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu
165 170 175Ser Thr Asn Leu Gln Glu Ser
Leu Arg Ser Lys Glu 180 18517564DNAHomo
sapiens 17atgaccaaca agtgtctcct ccaaattgct ctcctgttgt gcttctccac
tacagctctt 60tccatgagct acaacttgct tggattccta caaagaagca gcaattttca
gtgtcagaag 120ctcctgtggc aattgaatgg gaggcttgaa tactgcctca aggacaggat
gaactttgac 180atccctgagg agattaagca gctgcagcag ttccagaagg aggacgccgc
attgaccatc 240tatgagatgc tccagaacat ctttgctatt ttcagacaag attcatctag
cactggctgg 300aatgagacta ttgttgagaa cctcctggct aatgtctatc atcagataaa
ccatctgaag 360acagtcctgg aagaaaaact ggagaaagaa gatttcacca ggggaaaact
catgagcagt 420ctgcacctga aaagatatta tgggaggatt ctgcattacc tgaaggccaa
ggagtacagt 480cactgtgcct ggaccatagt cagagtggaa atcctaagga acttttactt
cattaacaga 540cttacaggtt acctccgaaa ctga
56418187PRTHomo sapiens 18Met Thr Asn Lys Cys Leu Leu Gln Ile
Ala Leu Leu Leu Cys Phe Ser1 5 10
15Thr Thr Ala Leu Ser Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln
Arg 20 25 30Ser Ser Asn Phe
Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg 35
40 45Leu Glu Tyr Cys Leu Lys Asp Arg Met Asn Phe Asp
Ile Pro Glu Glu 50 55 60Ile Lys Gln
Leu Gln Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile65 70
75 80Tyr Glu Met Leu Gln Asn Ile Phe
Ala Ile Phe Arg Gln Asp Ser Ser 85 90
95Ser Thr Gly Trp Asn Glu Thr Ile Val Glu Asn Leu Leu Ala
Asn Val 100 105 110Tyr His Gln
Ile Asn His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu 115
120 125Lys Glu Asp Phe Thr Arg Gly Lys Leu Met Ser
Ser Leu His Leu Lys 130 135 140Arg Tyr
Tyr Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser145
150 155 160His Cys Ala Trp Thr Ile Val
Arg Val Glu Ile Leu Arg Asn Phe Tyr 165
170 175Phe Ile Asn Arg Leu Thr Gly Tyr Leu Arg Asn
180 18519588DNAHomo sapiens 19atggccctcc tgttccctct
actggcagcc ctagtgatga ccagctatag ccctgttgga 60tctctgggct gtgatctgcc
tcagaaccat ggcctactta gcaggaacac cttggtgctt 120ctgcaccaaa tgaggagaat
ctcccctttc ttgtgtctca aggacagaag agacttcagg 180ttcccccagg agatggtaaa
agggagccag ttgcagaagg cccatgtcat gtctgtcctc 240catgagatgc tgcagcagat
cttcagcctc ttccacacag agcgctcctc tgctgcctgg 300aacatgaccc tcctagacca
actccacact ggacttcatc agcaactgca acacctggag 360acctgcttgc tgcaggtagt
gggagaagga gaatctgctg gggcaattag cagccctgca 420ctgaccttga ggaggtactt
ccagggaatc cgtgtctacc tgaaagagaa gaaatacagc 480gactgtgcct gggaagttgt
cagaatggaa atcatgaaat ccttgttctt atcaacaaac 540atgcaagaaa gactgagaag
taaagataga gacctgggct catcttga 58820194PRTHomo sapiens
20Ala Leu Leu Phe Pro Leu Leu Ala Ala Leu Val Met Thr Ser Tyr Ser1
5 10 15Pro Val Gly Ser Leu Gly
Cys Asp Leu Pro Gln Asn His Gly Leu Leu 20 25
30Ser Arg Asn Thr Leu Val Leu Leu His Gln Met Arg Arg
Ile Ser Pro 35 40 45Phe Leu Cys
Leu Lys Asp Arg Arg Asp Phe Arg Phe Pro Gln Glu Met 50
55 60Val Lys Gly Ser Gln Leu Gln Lys Ala His Val Met
Ser Val Leu His65 70 75
80Glu Met Leu Gln Gln Ile Phe Ser Leu Phe His Thr Glu Arg Ser Ser
85 90 95Ala Ala Trp Asn Met Thr
Leu Leu Asp Gln Leu His Thr Gly Leu His 100
105 110Gln Gln Leu Gln His Leu Glu Thr Cys Leu Leu Gln
Val Val Gly Glu 115 120 125Gly Glu
Ser Ala Gly Ala Ile Ser Ser Pro Ala Leu Thr Leu Arg Arg 130
135 140Tyr Phe Gln Gly Ile Arg Val Tyr Leu Lys Glu
Lys Lys Tyr Ser Asp145 150 155
160Cys Ala Trp Glu Val Val Arg Met Glu Ile Met Lys Ser Leu Phe Leu
165 170 175Ser Thr Asn Met
Gln Glu Arg Leu Arg Ser Lys Asp Arg Asp Leu Gly 180
185 190Ser Ser21501DNAHomo sapiens 21atgaaatata
caagttatat cttggctttt cagctctgca tcgttttggg ttctcttggc 60tgttactgcc
aggacccata tgtacaagaa gcagaaaacc ttaagaaata ttttaatgca 120ggtcattcag
atgtagcgga taatggaact cttttcttag gcattttgaa gaattggaaa 180gaggagagtg
acagaaaaat aatgcagagc caaattgtct ccttttactt caaacttttt 240aaaaacttta
aagatgacca gagcatccaa aagagtgtgg agaccatcaa ggaagacatg 300aatgtcaagt
ttttcaatag caacaaaaag aaacgagatg acttcgaaaa gctgactaat 360tattcggtaa
ctgacttgaa tgtccaacgc aaagcaatac atgaactcat ccaagtgatg 420gctgaactgt
cgccagcagc taaaacaggg aagcgaaaaa ggagtcagat gctgtttcga 480ggtcgaagag
catcccagta a 50122166PRTHomo
sapiens 22Met Lys Tyr Thr Ser Tyr Ile Leu Ala Phe Gln Leu Cys Ile Val
Leu1 5 10 15Gly Ser Leu
Gly Cys Tyr Cys Gln Asp Pro Tyr Val Gln Glu Ala Glu 20
25 30Asn Leu Lys Lys Tyr Phe Asn Ala Gly His
Ser Asp Val Ala Asp Asn 35 40
45Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp 50
55 60Arg Lys Ile Met Gln Ser Gln Ile Val
Ser Phe Tyr Phe Lys Leu Phe65 70 75
80Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln Lys Ser Val Glu
Thr Ile 85 90 95Lys Glu
Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg 100
105 110Asp Asp Phe Glu Lys Leu Thr Asn Tyr
Ser Val Thr Asp Leu Asn Val 115 120
125Gln Arg Lys Ala Ile His Glu Leu Ile Gln Val Met Ala Glu Leu Ser
130 135 140Pro Ala Ala Lys Thr Gly Lys
Arg Lys Arg Ser Gln Met Leu Phe Arg145 150
155 160Gly Arg Arg Ala Ser Gln
1652330DNAArtificial Sequencesynthesis 23gaattcatga agtgggtaac ctttatttcc
302425DNAArtificial
Sequencesynthesis 24catatgtgtg atctccctga gaccc
252525DNAArtificial Sequencesynthesis 25catatgtgtg
atctccctga gaccc
252627DNAArtificial Sequencesynthesis 26ggatccttac ttcctcctta atctttc
272725DNAArtificial Sequencesynthesis
27catatggcct tgacctttgc tttac
252826DNAArtificial Sequencesynthesis 28ggatcctcat tccttacttc ttaaac
262935DNAArtificial Sequencesynthesis
29tggcacagat gaggaaaatc tctcttttct cctgc
353036DNAArtificial Sequencesynthesis 30caggagaaaa gagagatttt cctcatctgt
gccagc 363122DNAArtificial
Sequencesynthesis 31catatgacca acaagtgtct cc
223224DNAArtificial Sequencesynthesis 32gaattctcag
tttcggaggt aacc
243325DNAArtificial Sequencesynthesis 33catatggccc tcctgttccc tctac
253426DNAArtificial Sequencesynthesis
34gaattctcaa gatgagccca ggtctc
263524DNAArtificial Sequencesynthesis 35catatgaaat atacaagtta tatc
243624DNAArtificial Sequencesynthesis
36gaattcttac tgggatgctc ttcg
243733DNAArtificial Sequencesynthesis 37ctgccttagg cttatgtgat ctccctgaga
ccc 333828DNAArtificial
Sequencesynthesis 38tctcgagtta cttcctcctt aatctttc
283933DNAArtificial Sequencesynthesis 39ctgccttagg
cttatgtgat ctgcctcaaa ccc
334027DNAArtificial Sequencesynthesis 40tctcgagtca ttccttactt cttaaac
274133DNAArtificial Sequencesynthesis
41ctgccttagg cttatacaac ttgcttggat tcc
334226DNAArtificial Sequencesynthesis 42cactcgagtc agtttcggag gtaacc
264337DNAArtificial Sequencesynthesis
43ctgccttagg cttatgtgat ctgcctcaga accatgg
374426DNAArtificial Sequencesynthesis 44ctcgagtcaa gatgagccca ggtctc
264537DNAArtificial Sequencesynthesis
45actccttagg cttacaggac ccatatgtac aagaagc
374624DNAArtificial Sequencesynthesis 46ctcgagttac tgggatgctc ttcg
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