Patent application title: Proteins with Improved Solubility and Methods for Producing and Using Same
Inventors:
Clifford C. Shone (Wiltshire, GB)
James A. Crawford (Dublin, OH, US)
IPC8 Class: AA61K39395FI
USPC Class:
4241671
Class name: Immunoglobulin, antiserum, antibody, or antibody fragment, except conjugate or complex of the same with nonimmunoglobulin material binds bacterium or component thereof or substance produced by said bacterium clostridium (e.g., clostridium tetani, etc.)
Publication date: 2009-10-29
Patent application number: 20090269358
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Patent application title: Proteins with Improved Solubility and Methods for Producing and Using Same
Inventors:
Clifford C. Shone
James A. Crawford
Agents:
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER;LLP
Assignees:
Origin: WASHINGTON, DC US
IPC8 Class: AA61K39395FI
USPC Class:
4241671
Patent application number: 20090269358
Abstract:
A method is provided for improving the solubility of proteins, for
example, bacterial toxins. In one embodiment, solubility is improved by
introducing point mutations that replace cysteine residues capable of
forming intermolecular disulfide bonds with other amino acid residues
that do not form such bonds. By abrogating the ability of the cysteine
residues to form inter-molecular disulfide bonds, aggregation of the
protein is reduced, thereby improving the solubility of the protein. In
another embodiment, solubility of the protein is improved by producing
truncated forms of the protein that express the LHN domain and a fragment
of the Hc domain. Proteins made according to the method of the invention
are useful, for example, as immunodiagnostic agents and vaccine
components.Claims:
1-34. (canceled)
35. A recombinant protein comprising a truncated botulinum serotype E toxin, wherein the truncation improves the solubility of the recombinant protein.
36. The protein of claim 35, wherein the truncation is in the Hc domain.
37. The protein of claim 35, wherein the truncated protein comprises the LHN/E domain and the amino terminal 103 amino acids of the Hc domain.
38. The protein of claim 35, wherein the truncated protein comprises the amino terminal 948 amino acids of the serotype E toxin.
39. The protein of claim 35, wherein the truncated protein comprises the LHN/E domain and the amino terminal 202 amino acids of the Hc domain.
40. The protein of claim 35, wherein the truncated protein comprises the amino terminal 1047 amino acids of the serotype E toxin.
41. The protein of claim 35, wherein the truncated protein comprises the LHN/E domain and the amino terminal 304 amino acids of the Hc domain.
42. The protein of claim 35, wherein the truncated protein comprises the amino terminal 1149 amino acids of the serotype E toxin.
43. A nucleic acid encoding a recombinant protein of claim 35.
44. A method for improving the solubility of a clostridial neurotoxin, comprising:(a) providing a nucleic acid sequence encoding a clostridial neurotoxin;(b) modifying the nucleic acid sequence so that it encodes the LHN fragment and a portion of the Hc fragment of the neurotoxin;(c) transforming the modified nucleic acid sequence into a host cell capable of expressing the modified nucleic acid sequence; and(d) expressing the modified nucleic acid sequence to produce the protein.
45. A method of treating or preventing botulism comprising administering a protein of claim 35 to a patient in need thereof.
46. A composition comprising a protein of claim 35 and a pharmaceutically acceptable carrier.
47. A method of protecting an individual from botulism, comprising administering to the individual a composition of claim 46.
48. A method of producing antibodies that neutralize a clostridial neurotoxin, comprising administering the composition of claim 46 to an animal, allowing the animal to develop neutralizing antibodies to the clostridial neurotoxin, and isolating an antiserum that neutralizes the clostridial neurotoxin from the animal.
49. An antiserum produced by the method of claim 48.
50. A method of treating exposure to a clostridial neurotoxin, comprising administering to a patient that has been exposed to the clostridial neurotoxin the antiserum of claim 49.
51. A mutated botulinum serotype E toxin comprising either or both of a leucine residue substituted for the tryptophan residue at position 1223 and a phenylalanine residue for the tyrosine residue at position 1224 of SEQ ID NO: 1 or SEQ ID NO: 2.
52. A method of treating or preventing botulism comprising administering a protein of claim 51 to a patient in need thereof.
53-58. (canceled)
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001]The present application claims the benefit of U.S. provisional application Nos. 60/724,274, filed Oct. 7, 2005, and 60/742,900, filed Dec. 7, 2005, the entire disclosures of which are incorporated by reference.
DESCRIPTION OF THE INVENTION
[0002]1. Field of the Invention
[0003]The present invention relates to methods for producing recombinant proteins that exhibit improved utility and process characteristics, such as solubility, compared to the corresponding native proteins. The invention also relates to proteins made according to the present methods, nucleic acids encoding the proteins, and the use of the proteins for prophylactic and therapeutic applications.
[0004]2, Background of the Invention
[0005]Proteins produced by organisms, and in particular microorganisms such as bacteria, are of interest because of their potential to serve as immunodiagnostic reagents, therapeutic agents, and vaccine components. Toxins are one group of proteins that have been extensively investigated for those purposes. It is often desirable, if not necessary, to purify proteins to remove contaminating materials that render them unsuitable for those uses. However, some proteins will form aggregates during purification. Aggregates tend to exhibit low solubility and other characteristics that are undesirable, such as low immunogenicity. In some cases, aggregates arise when cysteine residues in the protein of interest form aberrant inter-molecular and/or intra-molecular disulfide bonds.
[0006]One family of bacterial toxins of interest as immunodiagnostic reagents, therapeutic agents, and vaccine components are the clostridial neurotoxins, such as those from Clostridium botulinum and Clostridium butyricum. Botulinum neurotoxin (BoNT) is one of the most potent toxins known to man. Its ingestion or inhalation inhibits neurotransmitter release from synaptic vesicles, resulting in neuroparalysis and death. The use of Clostridium botulinum neurotoxins as vaccine components is disclosed in U.S. Pat. No. 5,919,665 to J. A. Williams, which is incorporated by reference into this application. In addition, U.S. Pat. Nos. 6,051,239 to Simpson et al., 6,287,566 to M. T. Dertzbaugh, and 6,461,617 to Shone et al., each of which is incorporated by reference into this application, disclose the use of fragments of clostridial neurotoxin as vaccine components.
[0007]Seven serologically distinct forms of clostridial neurotoxin exist: types A, B, C, D, E, F, and G. Full length neurotoxin type E, for example, is designated BoNT/E. Each neurotoxin type shares a common architecture in which a catalytic L-chain (LC, ˜50 kDa) is disulfide linked to a receptor binding and translocating H-chain (HC, ˜100 kDa). The HC polypeptide comprises all or part of two distinct functional domains. The carboxy-terminal half of the HC (˜50 kDa), termed the Hc domain, is involved in the high affinity, neurospecific binding of the neurotoxin to cell surface receptors on the target neuron. The amino-terminal half, termed the HN domain (˜50 kDa), mediates the translocation of at least some portion of the neurotoxin across cellular membranes such that the functional activity of the LC is expressed within the target cell. Although the heavy chain is required for BoNT to bind and enter the target cell, it is not toxic by itself.
[0008]One particular fragment of interest is the LHN fragment, such as the LHN fragment of neurotoxin E (LHN/E). LHN/E corresponds to the first 845 N-terminal amino acid residues of the full length botulinum (or butyricum) neurotoxin E. It includes the LC and HN domains. During in vivo expression, as well as during purification, both recombinant LHN/E (rLHN/E) and native forms of LHN/E form aggregates having a molecular mass ranging from about 120 kD to several million kD. Often LHN/E aggregates having masses of about 200 kD, 300 kD, 400 kD, 500 kD, 600 kD, 700 kD, and 800 kD are observed. Although aggregated rLHN/E can be recovered from insoluble lysate material by detergent extraction/reductant treatment and further purified (˜90%) by anion exchange (Q Sepharose) and gel filtration (Superdex 200) chromatography, the recovered aggregate has undesirable properties. For example, purified aggregated rLHN/E is recognized in a conformation sensitive ELISA to a much lesser degree (˜5-10-fold) compared to the native BoNT/E control, indicating that conformational epitopes are absent and/or buried within the aggregate. Further, animal efficacy data indicate that immunization with aggregated LHN/E does not protect animals against BoNT/E toxin challenge. Because conformational epitopes are known to play a key role in eliciting protective antibody responses, these results were not totally unexpected.
[0009]Different fermentation conditions, for example, slow initial growth, less potent inducers, and/or reduced induction temperatures, as well as different detergent extraction/reductant treatments and denaturation (e.g. urea)/refolding methodologies have been being explored in an effort to produce soluble, non-aggregated or less aggregated LHN/E, but with limited success. Hence, there is a need for protein toxins and toxin subfragments, such as LHN/E, that exhibit little or no aggregation and retain conformational epitopes that permit use of the toxins as immunodiagnostic reagents and vaccine components.
SUMMARY OF THE INVENTION
[0010]An object of the invention is to provide a method for reducing or preventing aggregate formation during purification and/or formulation of proteins, such as toxins and toxin fragments.
[0011]Another object of the invention is to provide greater batch-to-batch consistency within protein products when characterized by standard methods of protein analysis.
[0012]Still another object of the invention is to provide proteins with improved solubility and process characteristics.
[0013]Yet another object of the invention is to provide toxin proteins and toxin fragments with improved solubility and process characteristics for use as immunodiagnostic reagents, therapeutic agents, and vaccine components.
[0014]Additional objects and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by the compositions and methods particularly pointed out in the appended claims.
[0015]To achieve the objects and in accordance with the purpose of the invention, as embodied and broadly described herein, the disclosure describes in one embodiment a recombinant protein comprising at least one point mutation that substitutes a cysteine residue with another amino acid residue, wherein said substitution improves the solubility of the recombinant protein. In some embodiments, the protein is a toxin, such as a bacterial toxin, or a fragment thereof. The toxin may be a neurotoxin or neurotoxin fragment from, for example, Clostridium botulinum or Clostridium butyricum. In certain embodiments, the toxin is a clostridial neurotoxin, such as neurotoxin E, or a fragment thereof, such as an LHN/E fragment.
[0016]The disclosure also provides toxin fragments that are more soluble than certain other fragments. For example, in some embodiments, the toxin is a clostridial neurotoxin and the fragment is an LHN fragment that further comprises amino acid sequences from the Hc fragment, wherein the resulting LHN+HC fragment is more soluble than the LHN fragment. In certain embodiments, these fragments are recombinant neurotoxin E fragments.
[0017]The invention also comprises nucleic acids encoding the recombinant proteins set forth in the disclosure, vectors comprising those nucleic acid sequences, and methods of expressing the encoded proteins in host cells.
[0018]In yet other embodiments, the invention encompasses methods used in improving the solubility and process characteristics of the toxin and toxin fragments described in the specification.
[0019]The invention further comprises methods of using the disclosed toxin and toxin fragments as therapeutic agents and vaccine components.
[0020]Thus, the invention provides the following embodiments.
[0021]In one embodiment, then invention provides a recombinant protein comprising at least one point mutation that substitutes a cysteine residue with another amino acid residue, wherein said substitution improves the solubility of the recombinant protein.
[0022]In another embodiment, the protein is a toxin or non-toxic derivative of a toxin.
[0023]Still other embodiments of the invention encompass a toxin or non-toxic derivative of a toxin that is of bacterial origin.
[0024]In other embodiments, the bacterial toxin or toxin derivative is from either Clostridium botulinum or Clostridium butyricum.
[0025]In yet other embodiments, the toxin is a neurotoxin or neurotoxin derivative.
[0026]In still other embodiments, the neurotoxin is neurotoxin A, B, C, D, E, F, or G, or a non-toxic derivative thereof.
[0027]In yet other embodiments, the neurotoxin or non-toxic derivative is a fragment of neurotoxin E.
[0028]In other embodiments the fragment is the LHN/E fragment of neurotoxin E.
[0029]In still other embodiments, the neurotoxin E fragment comprises at least one cysteine to serine amino acid substitution.
[0030]In yet other embodiments, the substitution of serine for cysteine occurs at amino acid residue 26, amino acid residue 347, or both amino acid residue 26 and amino acid residue 347 compared to the LHN fragment of SEQ ID NO: 1 or SEQ ID NO: 2.
[0031]The invention includes embodiments in which a protein of the invention has active endopeptidase activity.
[0032]The invention also includes embodiments in which a protein of the invention has attenuated endopeptidase activity.
[0033]Among other embodiments, the invention also includes nucleic acids encoding a recombinant protein of the invention.
[0034]Similarly, other embodiments of the invention include a method for improving the solubility of a protein having at least one cysteine residue, that forms an intermolecular disulfide bond, comprising: [0035](a) providing a nucleic acid sequence encoding a recombinant protein comprising at least one cysteine residue; [0036](b) introducing at least one point mutation into the nucleic acid sequence that substitutes at least one cysteine residue with another amino acid residue; [0037](c) transforming a host cell with the mutated nucleic acid sequence; and [0038](d) expressing the nucleic acid sequence to produce the protein.
[0039]In some embodiments, the protein in the method is a toxin or non-toxic derivative thereof.
[0040]In other embodiments, the protein in the method is a toxin or non-toxic derivative of bacterial origin.
[0041]In yet other embodiments, the protein in the method is a bacterial toxin or toxin derivative from either Clostridium botulinum or Clostridium butyricum.
[0042]In still other embodiments, the protein in the method is a toxin a neurotoxin or neurotoxin derivative.
[0043]In some embodiments, the neurotoxin in the method is neurotoxin A, B, C, D, E, F, or G, or a non-toxic derivative thereof.
[0044]In other embodiments, the non-toxic derivative in the method is a is a fragment of neurotoxin E.
[0045]In yet other embodiments, the fragment in the method is the LHN/E fragment of neurotoxin E.
[0046]In some embodiments of the method, the amino acid introduced by the at least one point mutation is a serine.
[0047]In other embodiments of the method, the protein is a LHN fragment of clostridial neurotoxin E and the at least one point mutation substitutes a serine for a cysteine at amino acid residue 26, amino acid residue 347, or both amino acid residue 26 and amino acid residue 347 compared to the LHN fragment of SEQ ID NO: 1 or SEQ ID NO: 2.
[0048]In still other embodiments of the method, the point mutation is introduced by site-directed mutagenesis.
[0049]Various embodiments of the method further comprise isolating the protein.
[0050]In certain embodiments of the method, the host cell is a mammalian, plant, insect, fungal, or bacterial cell.
[0051]The methods of the invention include embodiments in which a protein of the invention has active endopeptidase activity.
[0052]The methods of the invention also include embodiments in which a protein of the invention has attenuated endopeptidase activity.
[0053]In some embodiments, the invention provides for the use of a protein of the invention for the manufacture of a medicament for the treatment or prevention of botulism.
[0054]Other embodiments of the invention include compositions comprising a protein of the invention and a pharmaceutically acceptable carrier.
[0055]In still other embodiments, the invention provides methods of protecting an individual from botulism, comprising administering to the individual a composition of the invention.
[0056]Yet other embodiments of the invention provide a method of producing antibodies that neutralize a clostridial neurotoxin, comprising administering a composition of the invention to an animal, allowing the animal to develop neutralizing antibodies to the clostridial neurotoxin, and isolating an antiserum that neutralizes the clostridial neurotoxin from the animal.
[0057]Other embodiments encompass an antiserum produced by a method of the invention.
[0058]In still other embodiments, the invention provides methods of treating exposure to a clostridial neurotoxin, comprising administering to a patient that has been exposed to the clostridial neurotoxin an antiserum of the invention.
[0059]In other embodiments, the invention provides a recombinant protein comprising a truncated botulinum serotype E toxin wherein the truncation improves the solubility of the recombinant protein.
[0060]In some embodiments, the protein comprises a truncation in the Hc domain.
[0061]In other embodiments, the truncated protein comprises the LHN/E domain and the amino terminal 103 amino acids of the Hc domain.
[0062]Still other embodiments of the invention encompass a truncated protein comprising the amino terminal 948 amino acids of the serotype E toxin.
[0063]In yet other embodiments, the truncated protein comprises the LHN/E domain and the amino terminal 202 amino acids of the Hc domain.
[0064]Still other embodiments of the invention encompass a truncated protein comprising the amino terminal 1047 amino acids of the serotype E toxin.
[0065]In other embodiments, the truncated protein comprises the LHN/E domain and the amino terminal 304 amino acids of the Hc domain.
[0066]Still other embodiments of the invention encompass a truncated protein comprising the amino terminal 1149 amino acids of the serotype E toxin.
[0067]Additional embodiments of the invention include nucleic acids encoding a truncated botulinum serotype E toxin.
[0068]In still other embodiments, the invention provides methods for improving the solubility of a clostridial neurotoxin, comprising: [0069](a) providing a nucleic acid sequence encoding clostridial neurotoxin; [0070](b) modifying the nucleic acid sequence so that it encodes the LHN fragment and a portion of the Hc fragment of the neurotoxin; [0071](c) transforming the modified nucleic acid sequence into a host cell capable of expressing the modified nucleic acid sequence; and [0072](d) expressing the modified nucleic acid sequence to produce the protein.
[0073]In yet other embodiments, the invention provides for use of a truncated botulinum serotype E toxin for the manufacture of a medicament for the treatment or prevention of botulism.
[0074]In other embodiments, the invention provides a composition comprising a truncated botulinum serotype E toxin and a pharmaceutically acceptable carrier.
[0075]In still other embodiments, the invention provides a method of protecting an individual from botulism, comprising administering to the individual a composition comprising a truncated botulinum serotype E toxin and a pharmaceutically acceptable carrier.
[0076]Other embodiments of the invention include a method of producing antibodies that neutralize a clostridial neurotoxin, comprising administering a composition comprising a truncated botulinum serotype E toxin and a pharmaceutically acceptable carrier to an animal, allowing the animal to develop neutralizing antibodies to the clostridial neurotoxin, and isolating an antiserum that neutralizes the clostridial neurotoxin from the animal.
[0077]In still other embodiments, the invention provides an antiserum produced by the method of the preceding paragraph.
[0078]In yet other embodiment, the invention provides methods of treating exposure to a clostridial neurotoxin, comprising administering to a patient that has been exposed to the clostridial neurotoxin an antiserum of the invention.
[0079]In yet another embodiment, the invention provides a mutated botulinum serotype E toxin comprising either or both of a leucine residue substituted for the tryptophan residue at position 1223 and a phenylalanine residue for the tyrosine residue at position 1224 of SEQ ID NO: 1 or SEQ ID NO: 2.
[0080]The invention also provides, in additional embodiments, for the use of a protein of the preceding paragraph for the manufacture of a medicament for the treatment or prevention of botulism.
[0081]In still other embodiments, the invention provides an in vitro method for improving the solubility of a protein having at least one cysteine residue that forms an intermolecular disulfide bond, comprising: [0082](a) providing a nucleic acid sequence encoding a recombinant protein comprising at least one cysteine residue; [0083](b) introducing at least one point mutation into the nucleic acid sequence that substitutes at least one cysteine residue with another amino acid residue; [0084](c) transforming a host cell with the mutated nucleic acid sequence; and [0085](d) expressing the nucleic acid sequence to produce the protein.
[0086]Yet other embodiments of the invention encompass an in vitro method for improving the solubility of a clostridial neurotoxin, comprising: [0087](a) providing a nucleic acid sequence encoding a clostridial neurotoxin; [0088](b) modifying the nucleic acid sequence so that it encodes the LHN fragment and a portion of the Hc fragment of the neurotoxin; [0089](c) transforming the modified nucleic acid sequence into a host cell capable of expressing the modified nucleic acid sequence; and [0090](d) expressing the modified nucleic acid sequence to produce the protein.
[0091]In still other embodiments, the invention provides a method of producing antibodies that neutralize a clostridial neurotoxin, comprising isolating antibodies elicited by an inoculated polypeptide, wherein said polypeptide is a protein according to the invention.
[0092]Yet other embodiments of the invention provided for the use of an antiserum of the invention for the manufacture of a medicament for treating exposure to clostridial neurotoxin.
[0093]It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. The accompanying drawings illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0094]FIGS. 1A and 1B are a polyacrylamide gel and chromatogram, respectively, showing production of recombinant LHN/E in Escherichia coli as a high molecular weight aggregate.
[0095]FIGS. 2A and 2B demonstrate high levels of mutated recombinant LHN/E in Escherichia coli as non-aggregated proteins.
[0096]FIGS. 3A and 3B are Coomassie Blue stained gels demonstrating that the solubility of the LHN/E-Hc protein increases as the length of the Hc sequence included in the protein increases.
[0097]FIGS. 4A and 4B are western blots demonstrating that the solubility of the LHN/E-Hc protein increases as the length of the Hc sequence included in the protein increases.
[0098]FIGS. 5A and 5B are a Coomassie Blue stained gel and western blot analysis, respectively, of the solubility of the LHN/E and LHN/E-Hc406 proteins in the presence of the reducing agent DTT.
[0099]FIG. 6 is a western blot comparing protein levels in total lysate, the soluble fraction, and the insoluble fraction for LHN proteins comprising Cys to Ser replacements at positions 26 and 347, and having or lacking an Hc fragment.
DESCRIPTION OF THE EMBODIMENTS
[0100]The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited herein, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated documents or portions of documents defines a term that contradicts that term's definition in the application, this application controls.
[0101]The use of the singular includes the plural unless specifically stated otherwise. The word "a" or "an" means "at least one" unless specifically stated otherwise. The use of "or" means "and/or" unless stated otherwise. The meaning of the phrase "at least one" is equivalent to the meaning of the phrase "one or more." Furthermore, the use of the term "including," as well as other forms, such as "includes" and "included," is not limiting. Also, terms such as "element" or "component" encompass both elements or components comprising one unit and elements or components comprising more than one unit unless specifically stated otherwise.
[0102]Neurotoxic proteins and fragments of these proteins are important immunodiagnostic reagents, therapeutic agents, and vaccine components. Functional neurotoxins are hazardous to work with, however, so investigators prefer to use recombinant proteins that have been genetically modified to reduce or eliminate their neurotoxicity. Unfortunately, it can be difficult to purify some of the recombinant, non-toxic, proteins because they often form aggregates, which have reduced solubility and are less effective reagents for use in immunodiagnostic, therapeutic, and vaccine applications. For example, although aggregated rLHN/E can be purified, it is recognized in a conformation-sensitive ELISA to a much lesser degree (˜5-10-fold) than is the native BoNT/E toxin, indicating that conformational epitopes are absent and/or buried within the aggregate. Also, immunization with aggregated LHN/E does not protect animals against BoNT/E toxin challenge.
[0103]Accordingly, the disclosure provides recombinant proteins with improved solubility. For example, in one embodiment, the disclosure describes a recombinant protein comprising at least one point mutation that substitutes, a cysteine residue with another amino acid residue, wherein said substitution improves the solubility of the recombinant protein. In some embodiments, the protein is a toxin, such as a bacterial toxin, for example, a neurotoxin or neurotoxin fragment from Clostridium botulinum or Clostridium butyricum. In some embodiments, the protein is a fragment of a neurotoxin, such as an LHN fragment, for example, an LHN/E fragment. In certain embodiments, the first and third cysteine residues, counting from the amino terminus of a naturally-occurring neurotoxin amino acid sequence, have been replaced with non-cysteine amino acids, such as serine, in the recombinant neurotoxin protein or fragment thereof. Although mutations in clostridial neurotoxin E are exemplified, clostridial neurotoxins A, B, C, D, F, and G can be modified in the same manner.
[0104]Nucleic acid sequences encoding various neurotoxins have been cloned and those nucleic acid sequences are known in the art. For example, a nucleic acid sequence of a full length neurotoxin E from C. botulinum is provided in GenBank accession no. AB082519. A nucleic acid sequence of a full length neurotoxin E from C. butyricum is provided in GenBank accession no. AB088207.
[0105]An example of an amino acid sequence of C. botulinum BoNT/E neurotoxin is:
TABLE-US-00001 (SEQ ID NO: 1) MPKINSFNYN DPVNDRTILY IKPGGCQEFY KSFNIMKNIW IIPERNVIGT TPQDFHPPTS LKNGDSSYYD PNYLQSDEEK DRFLKIVTKI FNRINNNLSG GILLEELSKA NPYLGNDNTP DNQFHIGDAS AVEIKFSNGS QDILLPNVII MGAEPDLFET NSSNISLRNN YMPSNHRFGS IAIVTFSPEY SFRFNDNCMN EFIQDPALTL MHELINSLHG LYGAKGITTK YTITQKQNPL ITNIRGTNIE EFLTFGGTDL NIITSAQSND IYTNLLADYK KIASKLSKVQ VSNPLLNPYK DVFEAKYGLD KDASGIYSVN INKFNDIFKK LYSFTEFDLR TKFQVKCRQT YIGQYKYFKL SNLLNDSIYN ISEGYNINNL KVNFRGQNAN LNPRIITPIT GRGLVKKIIR FCKNIVSVKG IRKSICIEIN NGELFFVASE NSYNDDNINT PKEIDDTVTS NNNYENDLDQ VILNFNSESA PGLSDEKLNL TIQNDAYIPK YDSNGTSDIE QHDVNELNVF FYLDAQKVPE GENNVNLTSS IDTALLEQPK IYTFFSSEFI NNVNKPVQAA LFVSWIQQVL VDFTTEANQK STVDKIADIS IVVPYIGLAL NIGNEAQKGN FKDALELLGA GILLEFEPEL LIPTILVFTI KSFLGSSDNK NKVIKAINNA LKERDEKWKE VYSFIVSNWM TKINTQFNKR KEQMYQALQN QVNAIKTIIE SKYNSYTLEE KNELTNKYDI KQIENELNQK VSIAMNNIDR FLTESSISYL MKIINEVKIN KLREYDENVK TYLLNYIIQH GSILGESQQE LNSMVTDTLN NSIPFKLSSY TDDKILISYF NKFFKRIKSS SVLNMRYKND KYVDTSGYDS NININGDVYK YPTNKNQFGI YNDKLSEVNI SQNDYIIYDN KYKNFSISFW VRIPNYDNKI VNVNNEYTII NCMRDNNSGW KVSLNHNEII WTFEDNRGIN QKLAFNYGNA NGISDYINKW IFVTITNDRL GDSKLYINGN LIDQKSILNL GNIHVSDNIL FKIVNCSYTR YIGIRYFNIF DKELDETEIQ TLYSNEPNTN ILKDFWGNYL LYDKEYYLLN VLKPNNFIDR RKDSTLSINN IRSTILLANR LYSGIKVKIQ RVNNSSTNDN LVRKNDQVYI NFVASKTHLF PLYADTATTN KEKTIKISSS GNRFNQVVVM NSVGNCTMNF KNNNGNNIGL LGFKADTVVA STWYYTHMRD HTNSNGCFWN FISEEHGWQE K.
[0106]This sequence includes the Met at residue 1. SEQ ID NO: 1 is the reference sequence for all numbering regarding C. botulinum BoNT/E and fragments thereof used in this specification, irrespective of whether those sequences have or do not have the first Met. The LHN/E fragment extends from the amino terminus to amino acid residue 845 (Lys) in SEQ ID NO: 1.
[0107]An example of an amino acid sequence of C. butryicum BoNT/E neurotoxin is:
TABLE-US-00002 (SEQ ID NO: 2) MPTINSFNYN DPVNNRTILY IKPGGCQQFY KSFNIMKNIW IIPERNVIGT IPQDFLPPTS LKNGDSSYYD PNYLQSDQEK DKFLKIVTKI FNRINDNLSG RILLEELSKA NPYLGNDNTP DGDFIINDAS AVPIQFSNGS QSILLPNVII MGAEPDLFET NSSNISLRNN YMPSNHGFGS IAIVTFSPEY SFRFKDNSMN EFIQDPALTL MHELIHSLHG LYGAKGITTK YTITQKQNPL ITNIRGTNIE EFLTFGGTDL NIITSAQSND IYTNLLADYK KIASKLSKVQ VSNPLLNPYK DVFEAKYGLD KDASGIYSVN INKFNDIFKK LYSFTEFDLA TKFQVKCRQT YIGQYKYFKL SNLLNDSIYN ISEGYNINNL KVNFRGQNAN LNPRIITPIT GRGLVKKIIR FCKNIVSVKG IRKSICIEIN NGELFFVASE NSYNDDNINT PKEIDDTVTS NNNYENDLDQ VILNFNSESA PGLSDEKLNL TIQNDAYIPK YDSNGTSDIE QHDVNELNVF FYLDAQKVPE GENNVNLTSS IDTALLEQPK IYTFFSSEFI NNVNKPVQAA LFVGWIQQVL VDFTTEANQK STVDKIADIS IVVPYIGLAL NIGNEAQKGN FKDALELLGA GILLEFEPEL LIPTILVFTI KSFLGSSDNK NKVIKAINNA LKERDEKWKE VYSFIVSNWM TKINTQFNKR KEQMYQALQN QVNALKAIIE SKYNSYTLEE KNELTNKYDI EQIENELNQK VSIAMNNIDR FLTESSISYL MKLINEVKIN KLREYDENVK TYLLDYIIKH GSILGESQQE LNSMVIDTLN NSIPFKLSSY TDDKILISYF NKFFKRIKSS SVLNNRYKND KYVDTSGYDS NININGDVYK YPTNKNQFGI YNDKLSEVNI SQNDYIIYDN KYKNFSISFW VRIPNYDNKI VNVNNEYTII NCMRDNNSGW KVSLNHNEII WTLQDNSGIN QKLAFNYGNA NGISDYINKW IFVTITNDRL GDSKLYINGN LIDKKSILNL GNIHVSDNIL FKIVNCSYTR YIGIRYFNIF DKELDETEIQ TLYNNEPNAN ILKDFWGNYL LYDKEYYLLN VLKPNNFINR RTDSTLSINN IRSTILLANR LYSGIKVKIQ RVNNSSTNDN LVRKNDQVYI NFVASKTHLL PLYADTATTN KEKTIKISSS GNRFNQVVVM NSVGNCTMNF KNNNGNNIGL LGFKADTVVA STWYYTHMRD NTNSNGFFWN FISEEHGWQE K.
[0108]This sequence includes the Met at residue 1. SEQ ID NO: 2 is the reference sequence for all numbering-regarding C. butyricum BoNT/E and fragments thereof used in this specification, irrespective of whether those sequences have or do not have the first Met. The LHN/E fragment extends from the amino terminus to amino acid residue 845 (Lys) in SEQ ID NO: 2.
[0109]In the case of neurotoxin proteins from C. botulinum and C. butyricum, any amino acid sequences disclosed in which the initial methionine is not included are also considered BoNT/E or LHN/E fragments, as appropriate, even though the numbering of the amino acid residues in that particular fragment assumes the Met at position 1.
[0110]BoNT/E and fragments thereof that have endopeptidase activity have a glutamate (E) at position 213 and a histidine (H) at position 216. The endopeptidase activity can be abolished by mutating these sequences. For example, the specification describes BoNT/E and fragments thereof in which the glutamate is replaced with glutamine (Q) (i.e., E213Q) and the histidine is replaced with a tyrosine (Y) (i.e., H216Y). These proteins lack endopeptidase activity.
[0111]Examples of recombinant proteins comprising at least one point mutation that substitutes a cysteine residue with another amino acid residue, wherein said substitution improves the solubility of the recombinant protein include, but are not limited to: [0112]a) a protein comprising residues 2 to 845 of the amino acid sequence set forth in SEQ ID NO: 1, wherein the cysteine at position 26 of SEQ ID NO: 1 is replaced with a serine; [0113]b) a protein comprising residues 2 to 845 of the amino acid sequence set forth in SEQ ID NO: 1, wherein the cysteine at position 347 of SEQ ID NO: 1 is replaced with a serine; [0114]c) a protein comprising residues 2 to 845 of the amino acid sequence set forth in SEQ ID NO: 1, wherein the cysteine at position 26 and the cysteine at position 347 of SEQ ID NO: 1 are each replaced with a serine; [0115]d) a protein comprising residues 2 to 845 of the amino acid sequence set forth in SEQ ID NO: 2, wherein the cysteine at position 26 of SEQ ID NO: 2 is replaced with a serine; [0116]e) a protein comprising residues 2 to 845 of the amino acid sequence set forth in SEQ ID NO: 2, wherein the cysteine at position 347 of SEQ ID NO: 2 is replaced with a serine; and [0117]f) a protein comprising residues 2 to 845 of the amino acid sequence set forth in SEQ ID NO: 2, wherein the cysteine at position 26 and the cysteine at position 347 of SEQ ID NO: 2 are each replaced with a serine.
[0118]In alternative embodiments, each of the proteins described in parts (a)-(f) of the preceding paragraph may consist of, rather than comprise, the respective amino acid sequences. Optionally, each of the proteins described in parts (a)-(f) of the preceding paragraph may further comprise a methionine at their respective amino termini. The solubility, immunogenicity, or both solubility and immunogenicity of the proteins described in parts (a)-(f) of the preceding paragraph is improved compared to proteins comprising or consisting of the corresponding amino acid sequence lacking the mentioned cysteine to serine replacement(s). In some embodiments, the proteins contain the E213Q and H216Y point mutations that abolish endopeptidase activity.
[0119]Additional examples of BoNT/E proteins or fragments thereof comprising Cys to Ser replacements are set forth in SEQ ID NOS: 7-14.
[0120]The disclosure also provides recombinant neurotoxins fragments that are more soluble than certain other fragments. For example, in some embodiments, the fragment is an LHN fragment that further comprises amino acid sequences from the Hc fragment, wherein the resulting LHN+Hc fragment is more soluble than the LHN fragment. In certain embodiments, the LHN fragment is an LHN/E fragment and the Hc fragment is an Hc/E fragment. The LHN fragment may optionally comprise at least one point mutation that substitutes a cysteine residue with another amino acid residue.
[0121]Examples of recombinant neurotoxins fragments that are more soluble than certain other fragments include, but are not limited to: [0122]a) a protein comprising residues 2 to 948 of the amino acid sequence set forth in SEQ ID NO: 1; [0123]b) a protein comprising residues 2 to 1047 of the amino acid sequence set forth in SEQ ID NO: 1; [0124]c) a protein comprising residues 2 to 1149 of the amino acid sequence set forth in SEQ ID NO: 1; [0125]d) a protein comprising residues 2 to 948 of the amino acid sequence set forth in SEQ ID NO: 2; [0126]e) a protein comprising residues 2 to 1047 of the amino acid sequence set forth in SEQ ID NO: 2; and [0127]f) a protein comprising residues 2 to 1149 of the amino acid sequence set forth in SEQ ID NO: 2.
[0128]In alternative embodiments, each of the proteins described in parts (a)-(f) of the preceding paragraph may consist of, rather than comprise, the respective amino acid sequences. Optionally, each of the proteins described in parts (a)-(f) of the preceding paragraph may further comprise a methionine at their respective amino terminus. The proteins described in parts (a)-(f) of the preceding paragraph may also optionally comprise a Cys to Ser substitution at amino acid residue 26, 347, or 26 and 347 of SEQ ID NO: 1, or SEQ ID NO: 2, as appropriate. The solubility, immunogenicity, or both solubility and immunogenicity of the proteins described in parts (a)-(f) of the preceding paragraph is improved compared to proteins consisting of amino acids 2-845 of the corresponding amino acid sequence. That is, the proteins described in parts (a)-(f) of the preceding paragraph have improved solubility, improved immunogenicity, or improved solubility and improved immunogenicity compared to the LHN fragment of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the proteins contain the E213Q and H216Y point mutations that abolish endopeptidase activity.
[0129]Additional examples of proteins comprising an extended LHN fragment are set forth in SEQ ID NO: 10 and SEQ ID NO: 14. Expression constructs LHN/E-Hc103; LHN/E-Hc202; LHN/E-Hc304; and LHN/E-Hc406 also contain nucleic acid sequences encoding proteins comprising an extended LHN fragment.
[0130]Proteins that comprise amino acid residues 1223 or 1224 (relative to SEQ ID NO: 1 or SEQ ID NO: 2) of neurotoxin E may further comprise an amino acid substitution at either residue 1223, residue 1224, or both residue 1223 and 1224. For example, the protein may comprise a tryptophan (W) to leucine (L) mutation at positions 1223 (i.e., W1223L), a tyrosine (Y) to phenylalanine (F) mutation at position 1224 (i.e., Y1224F), or a W1223L and a Y1224F double mutation. Examples of protein comprising these mutations are set forth in SEQ ID NO: 9 and SEQ ID NO: 13.
[0131]The invention also comprises nucleic acids encoding the various recombinant proteins described in the specification, vectors comprising those nucleic acid sequences, and methods of expressing the encoded protein in a host cell. Thus, in some embodiments, the nucleic acids encode modified C. botulinum or C. butyricum neurotoxins, such as neurotoxin E, or fragments thereof, such as an LHN fragment, that have improved solubility, immunogenicity, or both improved solubility and immunogenicity compared to the corresponding unmodified neurotoxin or neurotoxin fragment. Methods of measuring solubility and immunogenicity are known in the art, and include, but are not limited to, the methods described in the Examples.
[0132]An improvement in solubility can be accomplished by changing codons in the nucleic acid sequence that code for the amino acid cysteine to another amino acid that does not form a disulfide bond. Alternatively, or in addition, solubility can be improved by extending the sequence of a fragment, such as an LHN fragment of a clostridial neurotoxin, by providing additional sequences from an adjoining segment, such as an Hc fragment of a clostridial neurotoxin.
[0133]Methods of manipulating nucleic acids and of expressing the encoded proteins are known in the art, and include those described in Molecular Cloning, A Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor) and Current Protocols in Molecular Biology (Eds. Aufubel, Brent, Kingston, More, Feidman, Smith and Stuhl. Greene Publ. Assoc., Wiley-Interscience, N.Y., 1992). Thus, it is possible to modify a nucleic acid sequence by replacing the codon for cysteine with a codon for another amino acid. In general, a cysteine is replaced with a serine, but other amino acid substitutions are also possible, such as replacement of cysteine with alanine, glycine, valine, leucine, isoleucine, or modified forms of these amino acids, so long as the replacement amino acid does not readily form disulfide bonds. Alternatively, the cysteine residue may simply be deleted from the sequence. Obviously, a deletion must remove the codon for the cysteine from the nucleic acid sequence without introducing a frameshift. Techniques for making substitution and deletion mutations at predetermined sites in a nucleic acid having a known sequence are well known and include, but are not limited to, primer mutagenesis and other forms of site-directed mutagenesis.
[0134]Similarly, methods of joining two sequence fragments, such as an LHN and an Hc fragment of a clostridial neurotoxin, and of truncating a sequence, are known in the art. These include, but are not limited to, PCR-based techniques and techniques for ligating together two or more nucleic acid sequences.
[0135]Certain methods of expressing proteins are described in the Examples. Other methods can also be used, however. Generally, in order to express a protein, such as a bacterial toxin or fragment thereof, a suitable cell line is transformed with a DNA sequence encoding that protein under the control of known regulatory sequences. The transformed host cells are cultured and the protein recovered and isolated from the culture medium. The isolated expressed proteins are substantially free from other proteins with which they are co-produced as well as from other contaminants. Suitable cells or cell lines may be mammalian cells, such as Chinese hamster ovary cells (CHO), the monkey kidney COS-1 cell line, or mammalian CV-1 cells. The selection of suitable mammalian host cells and methods for transformation, culturing, amplification, screening, product production and purification are known in the art. (See, e.g., Gething and Sambrook, Nature, 293:620-625 (1981); Kaufman et al., Mol Cell Biol., 5(7):1750-1759 (1985); Howley et al., U.S. Pat. No. 4,419,446.))
[0136]Bacterial cells may also be used as suitable hosts for expression of a bacterial toxin or fragment thereof. For example, various strains of E. coli (e.g., HB101, MC1061) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas, other bacilli and the like may also be used. For expression of a protein in bacterial cells, DNA encoding the propeptide is generally not necessary.
[0137]In some embodiments, the bacterial toxin or fragment thereof is expressed using a vector that contains a DNA sequence encoding the protein and appropriate expression control sequences. Expression control sequences for such vectors are known to those skilled in the art and may be selected depending upon the host cells. In other embodiments, the bacterial toxin or fragment thereof is expressed as a fusion protein comprising the protein sequence of the bacterial toxin or fragment thereof and, for example, a tag to stabilize the resulting fusion protein or to simplify purification of the bacterial toxin or fragment thereof. Such tags are known in the art. Representative examples include sequences which encode a series of histidine residues, the epitope tag FLAG, the Herpes simplex glycoprotein D, beta-galactosidase, maltose binding protein, streptavidin tag or glutathione S-transferase.
[0138]The invention also encompasses the methods used for improving the solubility and process characteristics of a protein. For example, in some embodiments, the disclosure provides methods for improving the solubility and process characteristics of a protein having at least one cysteine residue that forms an intermolecular disulfide bond, comprising: [0139](a) providing a nucleic acid sequence encoding a recombinant protein comprising at least one cysteine residue; [0140](b) introducing at least one point mutation into the nucleic acid sequence that substitutes at least one cysteine residue with another amino acid residue; [0141](c) transforming a host cell with the mutated nucleic acid sequence; and [0142](d) expressing the nucleic acid sequence to produce the protein.
[0143]In other embodiments, the method comprises improving the solubility of a botulinum neurotoxin, comprising: [0144](a) providing a nucleic acid sequence encoding a botulinum neurotoxin; [0145](b) modifying the nucleic acid sequence so that it encodes the LHN fragment and a portion of the Hc fragment of the neurotoxin; [0146](c) transforming the modified nucleic acid sequence into a host cell capable of expressing the modified nucleic acid sequence; and [0147](d) expressing the modified nucleic acid sequence to produce the protein.
[0148]The methods for improving the solubility of a protein, such as a botulinum neurotoxin, can be entirely in vitro methods. In other embodiments, as discussed herein, the methods can include an in vivo aspect, such expressing the nucleic acid in vivo.
[0149]Unless otherwise stated, a "soluble" recombinant protein is one that is exists in solution in the cytoplasm of the host cell. If the protein contains a signal sequence the soluble protein is exported to the periplasmic space in bacteria hosts and is secreted into the culture medium in eukaryotic cells capable of secretion or by bacterial host possessing the appropriate genes. In contrast, an insoluble protein is one which exists in denatured form inside cytoplasmic granules (called an inclusion bodies) in the host cell. A soluble protein is a protein which is not found in an inclusion body inside the host cell or is found both in the cytoplasm and in inclusion bodies and in this case the protein may be present at high or low levels in the cytoplasm.
[0150]A soluble protein is distinct from a "solubilized" protein. An insoluble recombinant protein found inside an inclusion body may be solubilized (i.e., rendered into a soluble form) by treating purified inclusion bodies with denaturants such as guanidine hydrochloride, urea or sodium dodecyl sulfate (SDS). These denaturants must then be removed from the solubilized protein preparation to allow the recovered protein to renature (refold). A distinction is also made between proteins that are soluble (i.e., dissolved) in a solution devoid of significant amounts of ionic detergents (e.g., SDS) or denaturants (e.g., urea, guanidine hydrochloride) and proteins that exist as a suspension of insoluble protein molecules dispersed within the solution. A soluble protein will not be removed from a solution containing the protein by centrifugation using conditions sufficient to remove bacteria present in a liquid medium (e.g., centrifugation at 5,000 g for 4-5 minutes). A method of testing whether a protein is soluble or insoluble is described in U.S. Pat. No. 5,919,665, which is incorporated by reference.
[0151]The invention further encompasses methods of using the disclosed toxin and toxin fragments as therapeutic agents and vaccine components. Optionally, the disclosed toxin and toxin fragments are tested to ensure that they are free or substantially free of endotoxin activity. Methods of testing for endotoxin activity are known in the art.
[0152]Toxins and toxin fragments useful in vaccine compositions are those that can stimulate an antibody response that neutralizes a wild-type toxin of the same type. For example, when the toxin or toxin fragment is derived from clostridial type E neurotoxin, then the toxin or toxin fragment composition stimulates antibodies that neutralize the toxin activity of wild-type BoNT/E. By way of example only, one method for selecting clostridial neurotoxin toxins or neurotoxin fragments that can stimulate an antibody response that neutralizes wild-type BoNT activity is to determine whether the clostridial neurotoxin or neurotoxin fragment is immunoreactive with polyclonal neutralizing antibodies to wild-type BoNT of same type, such as BoNT/E. Methods of determining whether clostridial neurotoxin or neurotoxin fragment immunoreact with antibodies to wild-type BoNT include ELISA, western blot, double immunodiffusion assay, RIA, and the like. Another exemplary method comprises using the clostridial neurotoxin or neurotoxin fragments as an immunogen in mice, then determining whether the mice are protected from challenge with wild-type BoNT, such as wild-type BoNT/E.
[0153]A toxin or toxin fragment can be combined with a pharmaceutically acceptable carrier. Physiologically acceptable diluents include physiological saline solutions, and buffered saline solutions at neutral pH such as phosphate buffered saline. Other types of physiological carriers include liposomes and polymers. Optionally, the toxin or toxin fragments can be combined with an adjuvant. In some embodiments, the adjuvant is IC31®, produced by Intercell AG, Vienna, Austria. (See EP 1 326 634B and EP 1 296 713B.) In other embodiments, the adjuvant is a Toll-like receptor (TLR) agonist, such as a TLR 4 agonist, a TLR7 agonist, or a TLR9 agonist. TLR9 agonists include, for example, immunostimulatory CpG nucleic acid sequences. Other examples of adjuvants that can be used include, but are not limited to, Freund's incomplete adjuvant, Freund's complete adjuvant, alum, monophosphoryl lipid A, alum phosphate or hydroxide, and QS-21.
[0154]For vaccine formulations, the toxins or toxin fragments can also be combined with immunomodulators, such as interleukins and interferons, for example IL-1, IL-12, and IFN-γ.
[0155]When the toxin or toxin fragment is a clostridial neurotoxin or neurotoxin fragment, multiple types of clostridial neurotoxin or neurotoxin fragments can be used together in a formulation, or a single type can be used alone. Thus, vaccine formulations and compositions include, but are not limited to, BoNT/E or a fragment of BoNT/E, such as LHN/E, including LHN/E that is mutated and LHN/E that is extended by the inclusion of amino acid sequences from the Hc fragments, either alone or in combination with wild-type, mutant, or fragments of one or more of clostridial neurotoxins type A, B, C, D, F, or G. Many vaccine formulations are known to those of skill in the art.
[0156]The toxin or toxin fragment is added to a vaccine formulation in an amount effective to stimulate a protective immune response in an animal challenged with wild-type toxin. Thus, in preparing a vaccine formulation, the toxin or toxin fragment is used for the manufacture of a medicament for the treatment or prevention of botulism. Generation of protective antibodies that neutralize the wild-type toxin can be measured by testing the ability of the vaccine to protect an animal, such as a mouse, from challenge with a lethal dose of wild-type toxin. The amounts of the toxin or toxin fragment in the vaccine composition that can form a protective immune response are generally about 0.1 μg to 100 mg per kg of body weight. In some cases, about 1 μg to about 1 mg/kg body weight is used. Often, about 1 μg to about 100 μg toxin or toxin fragment per kg of body weight will be sufficient to stimulate a protective immune response, such as protective antibodies. An amount of toxin or toxin fragment that stimulates a protective immune response is considered to be an "effective amount."
[0157]Depending upon the circumstances, such as the animal to be vaccinated, either a single or multiple doses of the vaccine composition are administered to provide protective immunity against the wild-type toxin. The vaccine composition can be administered to an animal in a variety of ways, including subcutaneously, intramuscularly, intravenously, intradermally, orally, intranasally, ocularly, and intraperitoneally.
[0158]Any animal that is susceptible to the wild-type toxin can be vaccinated with the toxin or toxin fragment in an immunostimulatory composition. Examples of animals susceptible to clostridial neurotoxins include, but are not limited to, rabbits, rodents, birds, horses, cattle, and humans. Accordingly, a vaccine composition comprising a clostridial neurotoxin or neurotoxin fragment, such as the clostridial neurotoxins and neurotoxin fragments described herein, can be used to protect rabbits, rodents, birds, horses, cattle, and humans, including infant humans, from botulism, or from one or more of the symptoms of botulism, such as diarrheal disease, paralysis (either mild or severe), or death.
[0159]Toxin and toxin fragments can also be used to prepare compositions comprising neutralizing antibodies that immunoreact with the wild-type toxin. The resulting antisera can be used for the manufacture of a medicament for treating exposure to clostridial neurotoxin. Thus, antibody compositions, such as the isolated antisera or antibodies purified therefrom, can be used as a passive immune serum to prevent or treat botulism in patients exposed to the wild-type toxin. In such cases, the patient is a human, including an infant, suspected of having come in contact with the toxin, or is a human, including an infant, who has had known contact with the toxin, but is not yet showing symptoms of exposure. The antibody composition can also be used in a method of treating to ameliorate symptoms in patients that are suffering from the presence of toxin in their body. When the toxin is a clostridial neurotoxin, the symptoms include diarrhea and paralysis.
[0160]Methods of preparing passive immune sera are known in the art. For example, a vaccine composition can be administered to an animal such as a horse or a human until a neutralizing antibody response to wild type toxin is generated. Neutralizing antibodies can then be harvested, purified, and administered to patients exposed to, or exhibiting symptoms of contact with, the toxin to thereby treat or prevent botulism. In some cases, the antibodies are not purified after harvesting. When the neutralizing antibodies are from humans, the antibody preparation will generally be free of viruses, such as HIV and hepatitis. Methods of preparing human antisera are known in the art, and include the methods used to prepare IVIg. The neutralizing antibodies can be administered intravenously, intramuscularly, intradermally, or subcutaneously. Antibiotic therapy can be used in conjunction. Dosages for neutralizing antibodies generally vary from about 1 mg to 1000 mg/kg. Often, they are administered at a dosage of about 50-200 mg/kg of body weight.
[0161]The invention will be further clarified by the following examples, which are intended to be purely exemplary of the invention and in no way limiting.
EXAMPLES
Example 1
Site-Directed Mutagenesis of LHN/E to Remove One or More Cysteine Residues
[0162]Endopeptidase-ablating mutations (E213Q and H216Y relative to SEQ ID NO: 1 and SEQ ID NO: 2) were introduced into the LHN/E coding sequence and the resulting cassettes cloned into plasmid vector pET26b. Various E. coli host strains were transformed and assessed for the ability to direct expression of a rLHN/E fragment. While high levels of target protein could be produced, recombinant LHN/E was expressed in all hosts as high molecular mass aggregate (FIGS. 1A and 1B). Those SDS-PAGE and gel filtration studies conducted under reducing and non-reducing conditions showed that LHN/E aggregation results, at least in part, from intermolecular disulfide bond formation.
[0163]It was hypothesized that aggregation could be due to the formation of cysteine-linked disulfide bonds between multiple LHN/E polypeptides. Molecular biology approaches were pursued to increase expression of soluble, non-aggregated rLHN/E protein. Using computational analyses of the BoNT/E catalytic domain crystal structure two surface-proximal cysteine residues (Cys26 and Cys347 in SEQ ID NO: 1 and SEQ ID NO: 2) have been identified that most likely participate in intermolecular disulfide-bond-mediated bridging. Those residues were targeted for mutagenesis, and the aggregation properties of the mutated proteins were assessed.
[0164]LHN/E was cloned into the expression vector pET26b (Novagen) and this plasmid clone was used for the mutagenesis procedure. Specifically, site directed mutagenesis (QuikChange II XL site-directed mutagenesis kit, Stratagene) was used to introduce two point mutations into the LHN/E gene that change codons 26 and 347 (relative to a nucleic acid sequence encoding SEQ ID NO: 1 or SEQ ID NO: 2) from cysteine to structurally similar serine. While it is preferable to substitute the cysteine residue with a structurally similar amino acid, any amino acid may be substituted as long as that amino acid is incapable of forming an intermolecular disulfide bond.
[0165]The primer names and sequences used for mutagenesis are shown, with the nucleotides responsible for the cysteine to serine changes underlined. For both mutations, a TGC codon (cysteine) was changed to an AGC codon (serine). These two primers were used for the C26S mutation: C26S-LhnEfor: 5'-GTATATTAAACCGGGCGGCAGCCAGGAGTTTTATAAA AGC-3' (SEQ ID NO: 3) and C26S-LhnErev: 5'-GCTTTTATAAAACTCCTGG CTGCCGCCCGGTTTAATATAC-3' (SEQ ID NO: 4). These two primers were used for the C347S mutation: C347S-LhnEfor: 5'-GTACCAAATTTCAGGTGA AGAGCCGCCAAACCTACATCG-3' (SEQ ID NO: 5) and C347S-LhnErev: 5'-CGATGTAGGTTTGGCGGCTCTTCACCTGAAATTTGGTAC-3' (SEQ ID NO: 6).
[0166]Clones of pET26b/LHN/E containing each single point mutation and both point mutations were made. The C26S single mutant, C347S single mutant, and C26S/C347S double mutant clones were expressed and analyzed for disulfide bond-mediated aggregation. As shown in FIGS. 2A and 2B, analysis of reduced and non-reduced samples revealed that intermolecular disulfide bonding was abolished in each of the LHN/E clones (C26S single mutant, C347S single mutant, and C26S, C347S double mutant), whereas intermolecular disulfide bond formation was observable for the parental LHN/E clone lacking the serine substitutions.
Example 2
Extending the LHN/E Fragment with Hc Sequence Improves Solubility
[0167]The solubility of clostridial neurotoxin proteins can also be enhanced by creating proteins in which an LHN domain, or a fragment of an LHN domain, is expressed along with amino acid sequence from an Hc domain.
[0168]Recombinant truncated forms of the botulinum serotype E toxin, such as the LHN fragments, have proven difficult to produce (express) and purify due to low solubility. Even at low concentrations, insoluble forms are often expressed in a non-native multimeric and aggregated state which renders them poor immunogens and unable to elicit protective levels of toxin neutralizing antibodies. To address this issue and enable the production of soluble (and possibly monomeric) protein, recombinant derivatives of the LHN/E protein have been produced that carry various lengths of the adjoining Hc domain.
[0169]The following expression constructs were tested for protein solubility in the E. coli strain ER2566: LHN/E; LHN/E-Hc103; LHN/E-Hc202; LHN/E-Hc304; and LHN/E-Hc406. Fifteen milliliters of LB media containing kanamycin (30 μg/mL) were inoculated with each strain containing the construct listed above. Inoculations were made directly from frozen glycerol stocks. These cultures grew at 37° C. overnight with shaking. The next morning, the overnight cultures were diluted into 1 L of 2×YT containing kanamycin (30 μg/mL) (15 mL into IL of 2×YT) in Fernbach flasks. The cultures shook at 37° C. for three hours. OD600 ranged from 0.7-0.9 for all of the cultures. The Fernbach flasks were moved into 20° C. incubators, IPTG was added to 1 mM final concentration, and the flasks shook at 20° C. for 4 hours.
[0170]The 1 L cultures were collected by centrifugation and cell pellets were resuspended in 200 ml of 50 mM Tris, pH 8.0, 25 mM EDTA, pH 8.0. The cell suspensions were disrupted using the microfluidizer. Thirty-five milliliters of each cell lysate was centrifuged at 10,000×g for 30 minutes. The supernatants containing soluble protein were transferred into clean tubes and the insoluble pellets were resuspended in 35 mL of 50 mM Tris, pH 8.0, 25 mM EDTA, pH 8.0.
[0171]Equal volumes of total cell lysate, and soluble and insoluble fractions, were prepared for SDS-PAGE by the addition of sample buffer and boiling for 5 minutes. Equal amounts of each sample were subjected to SDS-PAGE. One set of gels was stained using MicroWave Blue reagent. Another set was transferred to PVDF membrane and subjected to western blotting using antisera specific for BoNT/E obtained from the Health Protection Agency.
[0172]The LHN/E solubility is enhanced with the addition of amino acid residues from the Hc domain. This can be seen by comparing the amount of LHN/E-Hc in soluble versus insoluble fractions in the Coomassie stained gels of FIG. 3 and the western blots of FIG. 4. LHN/E, which is devoid of any Hc sequence, is detected predominately in the insoluble fraction. This is also observed for LHN/E-Hc103 and LHN/E-Hc202. However, the solubility of recombinant proteins containing longer segments of the Hc domains is greatly enhanced. This can be seen for LHN/E-Hc406, which fractionates predominately with the soluble fraction, and also with LHN/E-Hc304, which also displays enhanced solubility. These conclusions were confirmed by densitometry scans of the Coomassie stained gel.
[0173]We also compared the effect of treating LHN/E and LHN/E-Hc406 with the reducing agent DTT. FIG. 5A shows a Coomassie stained gel and FIG. 5B a western blot of the DTT treated (+DTT) and untreated (-DTT) samples. In both the presence and absence of DTT, more LHN/E-Hc406 was found in the soluble (S) than in the insoluble (I) fraction.
[0174]The addition of Hc sequence to the LHN/E fragment improves its solubility, as does the replacement of Cys 26 and Cys 347 with serine residues. We have also prepared expression constructs in which we combined these approaches. Following induction of protein expression, the pellet was harvested, then lysed and microfluidized. The lysate was separated into soluble and insoluble fractions by centrifugation. The levels of protein in the total lysate, soluble fraction, and insoluble fraction compared by running on a gel and western blotting with the anti-BoNT/E antisera. As shown in FIG. 6, the LHN/E-Hc406 (C26S, C347S) mutant, like the LHN/E (C26S, C347S) mutant, partitions predominantly to the soluble fraction.
Example 3
Immunogenicity of LHN/E Cys to Ser Fragments and LHN/E-Hc Fragments
[0175]Abolishing the ability of LHN/E to form aberrant intermolecular disulfide bonds by replacing cysteine residues with amino acids that do not form disulfide bonds and extending the LHN/E fragment with Hc sequence are two techniques that improve the yield of monomeric or less aggregated protein. These modifications will enhance the immunogenicity and protective efficacy of the LHN/E fragment and the enzymatic activity of non-endopeptidase ablated toxins and toxin subfragments.
[0176]Immunogenicity of the recombinant proteins is tested in mice. Mice are immunized either with 10 μg of a LHN/E protein in which one or more cysteine residues has been replaced with another amino acid that does not form disulfide bonds, with 10 μg of a LHN/E protein that has been extended by inclusion of Hc sequence, with 10 μg of inactivated BoNT/E, or with other proteins described in the Examples. The proteins are suspended in an adjuvant emulsion. Control mice are immunized with saline emulsified in adjuvant for use as negative controls. The mice are immunized i.p. four times at 2-week intervals. One week after the last immunization, the mice are bled and the serum is analyzed by immunoblot for the presence of specific antibody. ELISA is used to determine the titer of the antisera. Two weeks after the last immunization, each mouse is challenged i.p. with 2 lethal doses of BoNT/E. Four days after challenge, the mice are scored for survivors.
Example 4
Amino Acid Sequences Encoded by Certain Constructs
[0177]The following amino acid sequences are encoded by constructs in which the type E neurotoxin is from Clostridium botulinum. In the sequences, the mutated cysteine residues, which have been substituted with serine residues, are indicated in bold and underline.
[0178]1. LHN/E (endopeptidase active):
TABLE-US-00003 (SEQ ID NO: 7) PKINSFNYNDPVNDRTILYIKPGGSQEFYKSFNIMKNIWIIPERNVIGTT PQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGG ILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIM GAEPDLFETNSSNISLRNNYMPSNHRFGSIAIVTFSPEYSFRFNDNCMNE FIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEE FLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKD VFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLRTKFQVKSRQTY IGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITG RGLVKKIIRFCKNIVSVKGIRKSICIEINNGELFFVASENSYNDDNINTP KEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKY DSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKI YTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADISI VVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIK SFLGSSDNKNKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRK EQMYQALQNQVNAIKTIIESKYNSYTLEEKNELTNKYDIKQIENELNQKV SIANNNIDRFLTESSISYLMKIINEVKINKLREYDENVKTYLLNYIIQHG SILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYFNKFFK.
[0179]2. LHN/E (endopeptidase attenuated):
TABLE-US-00004 (SEQ ID NO: 8) PKINSFNYNDPVNDRTILYIKPGGSQEFYKSFNIMKNIWIIPERNVIGTT PQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGG ILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIM CAEPDLFETNSSNISLRNNYMPSNHRFGSIAIVTFSPEYSFRFNDNCMNE FIQDPALTLMHQLIYSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEE FLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKD VFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLRTKFQVKSRQTY IGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITG RGLVKKIIRFCKNIVSVKGIRKSICIEINNGELFFVASENSYNDDNINTP KEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKY DSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKI YTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADISI VVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIK SFLGSSDNKNKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRK EQMYQALQNQVNAIKTIIESKYNSYTLEEKNELTNKYDIKQIENELNQKV SIANNNIDRFLTESSISYLMKIINEVKINKLREYDENVKTYLLNYIIQHG SILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYFNKFFK.
[0180]3. BoNT/E neurotoxin (endopeptidase attenuated):
TABLE-US-00005 (SEQ ID NO: 9) PKINSFNYNDPVNDRTILYIKPGGSQEFYKSFNIMKNIWIIPERNVIGTT PQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGG ILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIM GAEPDLFETNSSNISLRNNYMPSNHRFGSIAIVTFSPEYSFRFNDNCMNE FIQDPALTLMHQLIYSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEE FLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKD VFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLRTKFQVKSRQTY IGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITG RGLVKKIIRFCKNIVSVKGIRKSICIEINNGELFFVASENSYNDDNINTP KEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKY DSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKI YTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADISI VVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIK SFLGSSDNKNKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRK EQMYQALQNQVNAIKTIIESKYNSYTLEEKNELTNKYDIKQIENELNQKV SIAMNNIDRFLTESSISYLMKIINEVKINKLREYDENVKTYLLNYIIQHG SILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYFNKFFKRIKSSS VLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNIS QNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWK VSLNHNEIIWTFEDNRGINQKLAFNYGNANGISDYINKWIFVTITNDRLG DSKLYINGNLIDQKSILNLGNIHVSDNILFKIVNCSYTRYIGIRYFNIFD KELDETEIQTLYSNEPNTNILKDFWGNYLLYDKEYYLLNVLKPNNFIDRR KDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDNLVRKNDQVYIN FVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNCTMNFK NNNGNNIGLLGFKADTVVASTLFYTHMRDHTNSNGCFWNFISEEHGWQE K.
[0181]4. Extended LHN/E neurotoxin (endopeptidase attenuated):
TABLE-US-00006 (SEQ ID NO: 10) PKINSFNYNDPVNDRTILYIKPGGSQEFYKSFNIMKNIWIIPERNVIGTT PQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGG ILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIM GAEPDLFETNSSNISLRNNYMPSNHRFCSIAIVTFSPEYSFRFNDNCMNE FIQDPALTLMHQLIYSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEE FLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKD VFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLRTKFQVKSRQTY IGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITG RGLVKKIIRFCKNIVSVKGIRKSICIEINNGELFFVASENSYNDDNINTP KEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKY DSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKI YTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADISI VVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIK SFLGSSDNKNKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRK EQMYQALQNQVNAIKTIIESKYNSYTLEEKNELTNKYDIKQIENELNQKV SIANNNIDRFLTESSISYLMKIINEVKINKLREYDENVKTYLLNYIIQHG SILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYFNKFFKRIKSSS VLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNIS QNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWK VSLNHNEIIWTFEDNRGINQKLAFNYGNANGISDYINKWIFVTITNDRLG DSKLYINGNLIDQKSILNLGNIHVSDNILFKIVNCSYTRYIGIRYFNIFD KELDETEIQTLYSNE.
[0182]The following amino acid sequences are encoded by constructs in which the type E neurotoxin is from Clostridium butyricum. In the sequences, the mutated cysteine residues, which have been substituted with serine residues, are indicated in bold and underline.
[0183]5. LHN/E (endopeptidase active):
TABLE-US-00007 (SEQ ID NO: 11) PTINSFNYNDPVNNRTILYIKPGGSQQFYKSFNIMKNIWIIPERNVIGTI PQDFLPPTSLKNGDSSYYDPNYLQSDQEKDKFLKIVTKIFNRINDNLSCR ILLEELSKANPYLGNDNTPDGDFIINDASAVPIQFSNGSQSILLPNVIIM GAEPDLFETNSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFKDNSMNE FIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEE FLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKD VFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLATKFQVKSRQTY IGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITG RGLVKKIIRFCKNIVSVKGIRKSICIEINNGELFFVASENSYNDDNINTP KEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKY DSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKI YTFFSSEFINNVNKPVQAALFVGWIQQVLVDFTTEANQKSTVDKIADISI VVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIK SFLGSSDNKNKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKPK EQMYQALQNQVNALKAIIESKYNSYTLEEKNELTNKYDIEQIENELNQKV SIAMNNIDRFLTESSISYLMKLINEVKINKLREYDENVKTYLLDYIIKHG SILGESQQELNSMVIDTLNNSIPFKLSSYTDDKILISYFNKFFK.
[0184]6. LHN/E (endopeptidase attenuated):
TABLE-US-00008 (SEQ ID NO: 12) PTINSFNYNDPVNNRTILYIKPGGSQQFYKSFNIMKNIWIIPERNVIGTI PQDFLPPTSLKNGDSSYYDPNYLQSDQEKDKFLKIVTKIFNRINDNLSGR ILLEELSKANPYLGNDNTPDGDFIINDASAVPIQFSNGSQSILLPNVIIM GAEPDLFETNSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFKDNSMNE FIQDPALTLMHQLIYSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEE FLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKD VFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLATKFQVKSRQTY IGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITG RGLVKKIIRFCKNIVSVKGIRKSICIEINNGELFFVASSNSYNDDNINTP KEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKY DSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKI YTFFSSEFININNKPVQAALFVGWIQQVLVDFTTEANQKSTVDKIADISI VVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIK SFLGSSDNKNKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRK EQMYQALQNQVNALKAIIESKYNSYTLEEKNELTNKYDIEQIENELNQKV SIAMNNIDRFLTESSISYLMKLINEVKINKLREYDENVKTYLLDYIIKHG SILGESQQELNSMVIDTLNNSIPFKLSSYTDDKILISYFNKFFK.
[0185]7. BoNT/E neurotoxin (endopeptidase attenuated):
TABLE-US-00009 (SEQ ID NO: 13) PTINSFNYNDPVNNRTILYIKPGGSQQFYKSFNIMKNIWIIPERNVIGTI PQDFLPPTSLKNGDSSYYDPNYLQSDQEKDKFLKIVTKIFNRINDNLSGR ILLEELSKANPYLGNDNTPDGDFIINDASAVPIQFSNGSQSILLPNVIIM GAEPDLFETNSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFKDNSMNE FIQDPALTLMHQLIYSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEE FLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKD VFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLATKFQVKSRQTY IGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITG RGLVKKIIRFCKNIVSVKGIRKSICIEINKGELFFVASENSYNDDNINTP KEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKY DSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKI YTFFSSEFINNVNKPVQAALFVGWIQQVLVDFTTEANQKSTVDKIADISI VVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIK SFLGSSDNKNKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRK EQMYQALQNQVNALKAIIESKYNSYTLEEKNELTNKYDIEQIENELNQKV SIANNNIDRFLTESSISYLMKLINEVKINKLREYDENVKTYLLDYIIKHG SILGESQQELNSMVIDTLNNSIPFKLSSYTDDKILISYFNKFFKRIKSSS VLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNIS QNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWK VSLNHNEIIWTLQDNSGINQKLAFNYGNANGISDYINKWIFVTITNDRLG DSKLYINGNLIDKKSILNLGNIHVSDNILFKIVNCSYTRYIGIRYFNIFD KELDETEIQTLYNNEPNANILKDFWGNYLLYDKEYYLLNVLKPNNFINRR TDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDNLVRKNDQVYIN FVASKTHLLPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNCTMNFK NNNGNNIGLLGFKADTVVASTLFYTHNRDNTNSNGFFWNFISEEHGWQE K.
[0186]8. Extended LHN/E (endopeptidase attenuated):
TABLE-US-00010 (SEQ ID NO: 14) PKINSFNYNDPVNDRTILYIKPGGSQEFYKSFNIMKNIWIIPERNVIGTT PQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGG ILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIM GAEPDLFETNSSNISLRNNYMPSNHRFGSIAIVTFSPEYSFRFNDNCMNE FIQDPALTLMHQLIYSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEE FLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKD VFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLRTKFQVKSRQTY IGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITG RGLVKKIIRFCKNIVSVKGIRKSICIEINNGELFFVASENSYNDDNINTP KEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKY DSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKI YTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADISI VVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIK SFLGSSDNKNKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRK EQMYQALQNQVNAIKTIIESKYNSYTLEEKNELTNKYDIKQIENELNQKV SIANNNIDRFLTESSISYLMKIINEVKINKLREYDENVKTYLLNYIIQHG SILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYFNKFFKRIKSSS VLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNIS QNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWK VSLNHNEIIWTFEDNRGINQKLAFNYGNANGISDYINKWIFVTITNDRLG DSKLYINGNLIDQKSILNLGNIHVSDNILFKIVNCSYTRYIGIRYFNIFD KELDETEIQTLYSNE.
[0187]Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein, such as, for example, C. botulinum neurotoxins of type A, B, C, D, F, or G mutated or truncated according to the method of the invention that exhibit improved solubility. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.
Sequence CWU
1
1411251PRTClostridium botulinum 1Met Pro Lys Ile Asn Ser Phe Asn Tyr Asn
Asp Pro Val Asn Asp Arg1 5 10
15Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln Glu Phe Tyr Lys Ser20
25 30Phe Asn Ile Met Lys Asn Ile Trp Ile
Ile Pro Glu Arg Asn Val Ile35 40 45Gly
Thr Thr Pro Gln Asp Phe His Pro Pro Thr Ser Leu Lys Asn Gly50
55 60Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln
Ser Asp Glu Glu Lys65 70 75
80Asp Arg Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asn85
90 95Asn Leu Ser Gly Gly Ile Leu Leu Glu
Glu Leu Ser Lys Ala Asn Pro100 105 110Tyr
Leu Gly Asn Asp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp115
120 125Ala Ser Ala Val Glu Ile Lys Phe Ser Asn Gly
Ser Gln Asp Ile Leu130 135 140Leu Pro Asn
Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr145
150 155 160Asn Ser Ser Asn Ile Ser Leu
Arg Asn Asn Tyr Met Pro Ser Asn His165 170
175Arg Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe180
185 190Arg Phe Asn Asp Asn Cys Met Asn Glu
Phe Ile Gln Asp Pro Ala Leu195 200 205Thr
Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr Gly Ala210
215 220Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln
Lys Gln Asn Pro Leu225 230 235
240Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe
Gly245 250 255Gly Thr Asp Leu Asn Ile Ile
Thr Ser Ala Gln Ser Asn Asp Ile Tyr260 265
270Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys275
280 285Val Gln Val Ser Asn Pro Leu Leu Asn
Pro Tyr Lys Asp Val Phe Glu290 295 300Ala
Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn305
310 315 320Ile Asn Lys Phe Asn Asp
Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu325 330
335Phe Asp Leu Arg Thr Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr
Ile340 345 350Gly Gln Tyr Lys Tyr Phe Lys
Leu Ser Asn Leu Leu Asn Asp Ser Ile355 360
365Tyr Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe370
375 380Arg Gly Gln Asn Ala Asn Leu Asn Pro
Arg Ile Ile Thr Pro Ile Thr385 390 395
400Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn
Ile Val405 410 415Ser Val Lys Gly Ile Arg
Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly420 425
430Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn
Ile435 440 445Asn Thr Pro Lys Glu Ile Asp
Asp Thr Val Thr Ser Asn Asn Asn Tyr450 455
460Glu Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala465
470 475 480Pro Gly Leu Ser
Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala485 490
495Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu
Gln His500 505 510Asp Val Asn Glu Leu Asn
Val Phe Phe Tyr Leu Asp Ala Gln Lys Val515 520
525Pro Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr
Ala530 535 540Leu Leu Glu Gln Pro Lys Ile
Tyr Thr Phe Phe Ser Ser Glu Phe Ile545 550
555 560Asn Asn Val Asn Lys Pro Val Gln Ala Ala Leu Phe
Val Ser Trp Ile565 570 575Gln Gln Val Leu
Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr580 585
590Val Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile
Gly Leu595 600 605Ala Leu Asn Ile Gly Asn
Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala610 615
620Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro Glu
Leu625 630 635 640Leu Ile
Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser645
650 655Ser Asp Asn Lys Asn Lys Val Ile Lys Ala Ile Asn
Asn Ala Leu Lys660 665 670Glu Arg Asp Glu
Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn675 680
685Trp Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu
Gln Met690 695 700Tyr Gln Ala Leu Gln Asn
Gln Val Asn Ala Ile Lys Thr Ile Ile Glu705 710
715 720Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys
Asn Glu Leu Thr Asn725 730 735Lys Tyr Asp
Ile Lys Gln Ile Glu Asn Glu Leu Asn Gln Lys Val Ser740
745 750Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu
Ser Ser Ile Ser755 760 765Tyr Leu Met Lys
Ile Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu770 775
780Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu Asn Tyr Ile Ile
Gln His785 790 795 800Gly
Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Thr805
810 815Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu
Ser Ser Tyr Thr Asp820 825 830Asp Lys Ile
Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys835
840 845Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp
Lys Tyr Val Asp850 855 860Thr Ser Gly Tyr
Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys865 870
875 880Tyr Pro Thr Asn Lys Asn Gln Phe Gly
Ile Tyr Asn Asp Lys Leu Ser885 890 895Glu
Val Asn Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr900
905 910Lys Asn Phe Ser Ile Ser Phe Trp Val Arg Ile
Pro Asn Tyr Asp Asn915 920 925Lys Ile Val
Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg930
935 940Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His
Asn Glu Ile Ile945 950 955
960Trp Thr Phe Glu Asp Asn Arg Gly Ile Asn Gln Lys Leu Ala Phe Asn965
970 975Tyr Gly Asn Ala Asn Gly Ile Ser Asp
Tyr Ile Asn Lys Trp Ile Phe980 985 990Val
Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn995
1000 1005Gly Asn Leu Ile Asp Gln Lys Ser Ile Leu Asn
Leu Gly Asn Ile His1010 1015 1020Val Ser
Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg1025
1030 1035 1040Tyr Ile Gly Ile Arg Tyr Phe
Asn Ile Phe Asp Lys Glu Leu Asp Glu1045 1050
1055Thr Glu Ile Gln Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn Ile Leu1060
1065 1070Lys Asp Phe Trp Gly Asn Tyr Leu Leu
Tyr Asp Lys Glu Tyr Tyr Leu1075 1080
1085Leu Asn Val Leu Lys Pro Asn Asn Phe Ile Asp Arg Arg Lys Asp Ser1090
1095 1100Thr Leu Ser Ile Asn Asn Ile Arg Ser
Thr Ile Leu Leu Ala Asn Arg1105 1110 1115
1120Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn
Ser Ser1125 1130 1135Thr Asn Asp Asn Leu
Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe1140 1145
1150Val Ala Ser Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr Ala
Thr1155 1160 1165Thr Asn Lys Glu Lys Thr
Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe1170 1175
1180Asn Gln Val Val Val Met Asn Ser Val Gly Asn Cys Thr Met Asn
Phe1185 1190 1195 1200Lys Asn
Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp1205
1210 1215Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met
Arg Asp His Thr1220 1225 1230Asn Ser Asn
Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu His Gly Trp1235
1240 1245Gln Glu Lys125021251PRTClostridium butyricum 2Met
Pro Thr Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asn Arg1
5 10 15Thr Ile Leu Tyr Ile Lys Pro Gly
Gly Cys Gln Gln Phe Tyr Lys Ser20 25
30Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile35
40 45Gly Thr Ile Pro Gln Asp Phe Leu Pro Pro
Thr Ser Leu Lys Asn Gly50 55 60Asp Ser
Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Gln Glu Lys65
70 75 80Asp Lys Phe Leu Lys Ile Val
Thr Lys Ile Phe Asn Arg Ile Asn Asp85 90
95Asn Leu Ser Gly Arg Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro100
105 110Tyr Leu Gly Asn Asp Asn Thr Pro Asp
Gly Asp Phe Ile Ile Asn Asp115 120 125Ala
Ser Ala Val Pro Ile Gln Phe Ser Asn Gly Ser Gln Ser Ile Leu130
135 140Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro
Asp Leu Phe Glu Thr145 150 155
160Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn
His165 170 175Gly Phe Gly Ser Ile Ala Ile
Val Thr Phe Ser Pro Glu Tyr Ser Phe180 185
190Arg Phe Lys Asp Asn Ser Met Asn Glu Phe Ile Gln Asp Pro Ala Leu195
200 205Thr Leu Met His Glu Leu Ile His Ser
Leu His Gly Leu Tyr Gly Ala210 215 220Lys
Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu225
230 235 240Ile Thr Asn Ile Arg Gly
Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly245 250
255Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile
Tyr260 265 270Thr Asn Leu Leu Ala Asp Tyr
Lys Lys Ile Ala Ser Lys Leu Ser Lys275 280
285Val Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu290
295 300Ala Lys Tyr Gly Leu Asp Lys Asp Ala
Ser Gly Ile Tyr Ser Val Asn305 310 315
320Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe
Thr Glu325 330 335Phe Asp Leu Ala Thr Lys
Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile340 345
350Gly Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser
Ile355 360 365Tyr Asn Ile Ser Glu Gly Tyr
Asn Ile Asn Asn Leu Lys Val Asn Phe370 375
380Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr385
390 395 400Gly Arg Gly Leu
Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val405 410
415Ser Val Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn
Asn Gly420 425 430Glu Leu Phe Phe Val Ala
Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile435 440
445Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn
Tyr450 455 460Glu Asn Asp Leu Asp Gln Val
Ile Leu Asn Phe Asn Ser Glu Ser Ala465 470
475 480Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile
Gln Asn Asp Ala485 490 495Tyr Ile Pro Lys
Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln His500 505
510Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln
Lys Val515 520 525Pro Glu Gly Glu Asn Asn
Val Asn Leu Thr Ser Ser Ile Asp Thr Ala530 535
540Leu Leu Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe
Ile545 550 555 560Asn Asn
Val Asn Lys Pro Val Gln Ala Ala Leu Phe Val Gly Trp Ile565
570 575Gln Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn
Gln Lys Ser Thr580 585 590Val Asp Lys Ile
Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu595 600
605Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys
Asp Ala610 615 620Leu Glu Leu Leu Gly Ala
Gly Ile Leu Leu Glu Phe Glu Pro Glu Leu625 630
635 640Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys
Ser Phe Leu Gly Ser645 650 655Ser Asp Asn
Lys Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys660
665 670Glu Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe
Ile Val Ser Asn675 680 685Trp Met Thr Lys
Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met690 695
700Tyr Gln Ala Leu Gln Asn Gln Val Asn Ala Leu Lys Ala Ile
Ile Glu705 710 715 720Ser
Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn725
730 735Lys Tyr Asp Ile Glu Gln Ile Glu Asn Glu Leu
Asn Gln Lys Val Ser740 745 750Ile Ala Met
Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser755
760 765Tyr Leu Met Lys Leu Ile Asn Glu Val Lys Ile Asn
Lys Leu Arg Glu770 775 780Tyr Asp Glu Asn
Val Lys Thr Tyr Leu Leu Asp Tyr Ile Ile Lys His785 790
795 800Gly Ser Ile Leu Gly Glu Ser Gln Gln
Glu Leu Asn Ser Met Val Ile805 810 815Asp
Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp820
825 830Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe
Phe Lys Arg Ile Lys835 840 845Ser Ser Ser
Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp850
855 860Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly
Asp Val Tyr Lys865 870 875
880Tyr Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser885
890 895Glu Val Asn Ile Ser Gln Asn Asp Tyr
Ile Ile Tyr Asp Asn Lys Tyr900 905 910Lys
Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn915
920 925Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile
Ile Asn Cys Met Arg930 935 940Asp Asn Asn
Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile945
950 955 960Trp Thr Leu Gln Asp Asn Ser
Gly Ile Asn Gln Lys Leu Ala Phe Asn965 970
975Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe980
985 990Val Thr Ile Thr Asn Asp Arg Leu Gly
Asp Ser Lys Leu Tyr Ile Asn995 1000 1005Gly
Asn Leu Ile Asp Lys Lys Ser Ile Leu Asn Leu Gly Asn Ile His1010
1015 1020Val Ser Asp Asn Ile Leu Phe Lys Ile Val Asn
Cys Ser Tyr Thr Arg1025 1030 1035
1040Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp
Glu1045 1050 1055Thr Glu Ile Gln Thr Leu
Tyr Asn Asn Glu Pro Asn Ala Asn Ile Leu1060 1065
1070Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr
Leu1075 1080 1085Leu Asn Val Leu Lys Pro
Asn Asn Phe Ile Asn Arg Arg Thr Asp Ser1090 1095
1100Thr Leu Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn
Arg1105 1110 1115 1120Leu Tyr
Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser Ser1125
1130 1135Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gln Val
Tyr Ile Asn Phe1140 1145 1150Val Ala Ser
Lys Thr His Leu Leu Pro Leu Tyr Ala Asp Thr Ala Thr1155
1160 1165Thr Asn Lys Glu Lys Thr Ile Lys Ile Ser Ser Ser
Gly Asn Arg Phe1170 1175 1180Asn Gln Val
Val Val Met Asn Ser Val Gly Asn Cys Thr Met Asn Phe1185
1190 1195 1200Lys Asn Asn Asn Gly Asn Asn
Ile Gly Leu Leu Gly Phe Lys Ala Asp1205 1210
1215Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp Asn Thr1220
1225 1230Asn Ser Asn Gly Phe Phe Trp Asn Phe
Ile Ser Glu Glu His Gly Trp1235 1240
1245Gln Glu Lys1250340DNAArtificial SequenceDescription of Artificial
Sequence Synthetic Primer 3gtatattaaa ccgggcggca gccaggagtt
ttataaaagc 40440DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
4gcttttataa aactcctggc tgccgcccgg tttaatatac
40539DNAArtificial SequenceDescription of Artificial Sequence Synthetic
Primer 5gtaccaaatt tcaggtgaag agccgccaaa cctacatcg
39639DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 6cgatgtaggt ttggcggctc ttcacctgaa atttggtac
397844PRTClostridium botulinum 7Pro Lys Ile Asn Ser
Phe Asn Tyr Asn Asp Pro Val Asn Asp Arg Thr1 5
10 15Ile Leu Tyr Ile Lys Pro Gly Gly Ser Gln Glu Phe
Tyr Lys Ser Phe20 25 30Asn Ile Met Lys
Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile Gly35 40
45Thr Thr Pro Gln Asp Phe His Pro Pro Thr Ser Leu Lys Asn
Gly Asp50 55 60Ser Ser Tyr Tyr Asp Pro
Asn Tyr Leu Gln Ser Asp Glu Glu Lys Asp65 70
75 80Arg Phe Leu Lys Ile Val Thr Lys Ile Phe Asn
Arg Ile Asn Asn Asn85 90 95Leu Ser Gly
Gly Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro Tyr100
105 110Leu Gly Asn Asp Asn Thr Pro Asp Asn Gln Phe His
Ile Gly Asp Ala115 120 125Ser Ala Val Glu
Ile Lys Phe Ser Asn Gly Ser Gln Asp Ile Leu Leu130 135
140Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu
Thr Asn145 150 155 160Ser
Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His Arg165
170 175Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro
Glu Tyr Ser Phe Arg180 185 190Phe Asn Asp
Asn Cys Met Asn Glu Phe Ile Gln Asp Pro Ala Leu Thr195
200 205Leu Met His Glu Leu Ile His Ser Leu His Gly Leu
Tyr Gly Ala Lys210 215 220Gly Ile Thr Thr
Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu Ile225 230
235 240Thr Asn Ile Arg Gly Thr Asn Ile Glu
Glu Phe Leu Thr Phe Gly Gly245 250 255Thr
Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr Thr260
265 270Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser
Lys Leu Ser Lys Val275 280 285Gln Val Ser
Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu Ala290
295 300Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr
Ser Val Asn Ile305 310 315
320Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu Phe325
330 335Asp Leu Arg Thr Lys Phe Gln Val Lys
Ser Arg Gln Thr Tyr Ile Gly340 345 350Gln
Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile Tyr355
360 365Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu
Lys Val Asn Phe Arg370 375 380Gly Gln Asn
Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr Gly385
390 395 400Arg Gly Leu Val Lys Lys Ile
Ile Arg Phe Cys Lys Asn Ile Val Ser405 410
415Val Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly Glu420
425 430Leu Phe Phe Val Ala Ser Glu Asn Ser
Tyr Asn Asp Asp Asn Ile Asn435 440 445Thr
Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr Glu450
455 460Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn
Ser Glu Ser Ala Pro465 470 475
480Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala
Tyr485 490 495Ile Pro Lys Tyr Asp Ser Asn
Gly Thr Ser Asp Ile Glu Gln His Asp500 505
510Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln Lys Val Pro515
520 525Glu Gly Glu Asn Asn Val Asn Leu Thr
Ser Ser Ile Asp Thr Ala Leu530 535 540Leu
Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile Asn545
550 555 560Asn Val Asn Lys Pro Val
Gln Ala Ala Leu Phe Val Ser Trp Ile Gln565 570
575Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr
Val580 585 590Asp Lys Ile Ala Asp Ile Ser
Ile Val Val Pro Tyr Ile Gly Leu Ala595 600
605Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala Leu610
615 620Glu Leu Leu Gly Ala Gly Ile Leu Leu
Glu Phe Glu Pro Glu Leu Leu625 630 635
640Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly
Ser Ser645 650 655Asp Asn Lys Asn Lys Val
Ile Lys Ala Ile Asn Asn Ala Leu Lys Glu660 665
670Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn
Trp675 680 685Met Thr Lys Ile Asn Thr Gln
Phe Asn Lys Arg Lys Glu Gln Met Tyr690 695
700Gln Ala Leu Gln Asn Gln Val Asn Ala Ile Lys Thr Ile Ile Glu Ser705
710 715 720Lys Tyr Asn Ser
Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn Lys725 730
735Tyr Asp Ile Lys Gln Ile Glu Asn Glu Leu Asn Gln Lys Val
Ser Ile740 745 750Ala Met Asn Asn Ile Asp
Arg Phe Leu Thr Glu Ser Ser Ile Ser Tyr755 760
765Leu Met Lys Ile Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu
Tyr770 775 780Asp Glu Asn Val Lys Thr Tyr
Leu Leu Asn Tyr Ile Ile Gln His Gly785 790
795 800Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser
Met Val Thr Asp805 810 815Thr Leu Asn Asn
Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp Asp820 825
830Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys835
8408844PRTClostridium botulinum 8Pro Lys Ile Asn Ser Phe Asn Tyr
Asn Asp Pro Val Asn Asp Arg Thr1 5 10
15Ile Leu Tyr Ile Lys Pro Gly Gly Ser Gln Glu Phe Tyr Lys Ser
Phe20 25 30Asn Ile Met Lys Asn Ile Trp
Ile Ile Pro Glu Arg Asn Val Ile Gly35 40
45Thr Thr Pro Gln Asp Phe His Pro Pro Thr Ser Leu Lys Asn Gly Asp50
55 60Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln
Ser Asp Glu Glu Lys Asp65 70 75
80Arg Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asn
Asn85 90 95Leu Ser Gly Gly Ile Leu Leu
Glu Glu Leu Ser Lys Ala Asn Pro Tyr100 105
110Leu Gly Asn Asp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp Ala115
120 125Ser Ala Val Glu Ile Lys Phe Ser Asn
Gly Ser Gln Asp Ile Leu Leu130 135 140Pro
Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr Asn145
150 155 160Ser Ser Asn Ile Ser Leu
Arg Asn Asn Tyr Met Pro Ser Asn His Arg165 170
175Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe
Arg180 185 190Phe Asn Asp Asn Cys Met Asn
Glu Phe Ile Gln Asp Pro Ala Leu Thr195 200
205Leu Met His Gln Leu Ile Tyr Ser Leu His Gly Leu Tyr Gly Ala Lys210
215 220Gly Ile Thr Thr Lys Tyr Thr Ile Thr
Gln Lys Gln Asn Pro Leu Ile225 230 235
240Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe
Gly Gly245 250 255Thr Asp Leu Asn Ile Ile
Thr Ser Ala Gln Ser Asn Asp Ile Tyr Thr260 265
270Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys
Val275 280 285Gln Val Ser Asn Pro Leu Leu
Asn Pro Tyr Lys Asp Val Phe Glu Ala290 295
300Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn Ile305
310 315 320Asn Lys Phe Asn
Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu Phe325 330
335Asp Leu Arg Thr Lys Phe Gln Val Lys Ser Arg Gln Thr Tyr
Ile Gly340 345 350Gln Tyr Lys Tyr Phe Lys
Leu Ser Asn Leu Leu Asn Asp Ser Ile Tyr355 360
365Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe
Arg370 375 380Gly Gln Asn Ala Asn Leu Asn
Pro Arg Ile Ile Thr Pro Ile Thr Gly385 390
395 400Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys
Asn Ile Val Ser405 410 415Val Lys Gly Ile
Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly Glu420 425
430Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn
Ile Asn435 440 445Thr Pro Lys Glu Ile Asp
Asp Thr Val Thr Ser Asn Asn Asn Tyr Glu450 455
460Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala
Pro465 470 475 480Gly Leu
Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala Tyr485
490 495Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile
Glu Gln His Asp500 505 510Val Asn Glu Leu
Asn Val Phe Phe Tyr Leu Asp Ala Gln Lys Val Pro515 520
525Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr
Ala Leu530 535 540Leu Glu Gln Pro Lys Ile
Tyr Thr Phe Phe Ser Ser Glu Phe Ile Asn545 550
555 560Asn Val Asn Lys Pro Val Gln Ala Ala Leu Phe
Val Ser Trp Ile Gln565 570 575Gln Val Leu
Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr Val580
585 590Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr
Ile Gly Leu Ala595 600 605Leu Asn Ile Gly
Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala Leu610 615
620Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro Glu
Leu Leu625 630 635 640Ile
Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser Ser645
650 655Asp Asn Lys Asn Lys Val Ile Lys Ala Ile Asn
Asn Ala Leu Lys Glu660 665 670Arg Asp Glu
Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn Trp675
680 685Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys
Glu Gln Met Tyr690 695 700Gln Ala Leu Gln
Asn Gln Val Asn Ala Ile Lys Thr Ile Ile Glu Ser705 710
715 720Lys Tyr Asn Ser Tyr Thr Leu Glu Glu
Lys Asn Glu Leu Thr Asn Lys725 730 735Tyr
Asp Ile Lys Gln Ile Glu Asn Glu Leu Asn Gln Lys Val Ser Ile740
745 750Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu
Ser Ser Ile Ser Tyr755 760 765Leu Met Lys
Ile Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu Tyr770
775 780Asp Glu Asn Val Lys Thr Tyr Leu Leu Asn Tyr Ile
Ile Gln His Gly785 790 795
800Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Thr Asp805
810 815Thr Leu Asn Asn Ser Ile Pro Phe Lys
Leu Ser Ser Tyr Thr Asp Asp820 825 830Lys
Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys835
84091250PRTClostridium botulinum 9Pro Lys Ile Asn Ser Phe Asn Tyr Asn Asp
Pro Val Asn Asp Arg Thr1 5 10
15Ile Leu Tyr Ile Lys Pro Gly Gly Ser Gln Glu Phe Tyr Lys Ser Phe20
25 30Asn Ile Met Lys Asn Ile Trp Ile Ile
Pro Glu Arg Asn Val Ile Gly35 40 45Thr
Thr Pro Gln Asp Phe His Pro Pro Thr Ser Leu Lys Asn Gly Asp50
55 60Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser
Asp Glu Glu Lys Asp65 70 75
80Arg Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asn Asn85
90 95Leu Ser Gly Gly Ile Leu Leu Glu Glu
Leu Ser Lys Ala Asn Pro Tyr100 105 110Leu
Gly Asn Asp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp Ala115
120 125Ser Ala Val Glu Ile Lys Phe Ser Asn Gly Ser
Gln Asp Ile Leu Leu130 135 140Pro Asn Val
Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr Asn145
150 155 160Ser Ser Asn Ile Ser Leu Arg
Asn Asn Tyr Met Pro Ser Asn His Arg165 170
175Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe Arg180
185 190Phe Asn Asp Asn Cys Met Asn Glu Phe
Ile Gln Asp Pro Ala Leu Thr195 200 205Leu
Met His Gln Leu Ile Tyr Ser Leu His Gly Leu Tyr Gly Ala Lys210
215 220Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys
Gln Asn Pro Leu Ile225 230 235
240Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly
Gly245 250 255Thr Asp Leu Asn Ile Ile Thr
Ser Ala Gln Ser Asn Asp Ile Tyr Thr260 265
270Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys Val275
280 285Gln Val Ser Asn Pro Leu Leu Asn Pro
Tyr Lys Asp Val Phe Glu Ala290 295 300Lys
Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn Ile305
310 315 320Asn Lys Phe Asn Asp Ile
Phe Lys Lys Leu Tyr Ser Phe Thr Glu Phe325 330
335Asp Leu Arg Thr Lys Phe Gln Val Lys Ser Arg Gln Thr Tyr Ile
Gly340 345 350Gln Tyr Lys Tyr Phe Lys Leu
Ser Asn Leu Leu Asn Asp Ser Ile Tyr355 360
365Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe Arg370
375 380Gly Gln Asn Ala Asn Leu Asn Pro Arg
Ile Ile Thr Pro Ile Thr Gly385 390 395
400Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile
Val Ser405 410 415Val Lys Gly Ile Arg Lys
Ser Ile Cys Ile Glu Ile Asn Asn Gly Glu420 425
430Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile
Asn435 440 445Thr Pro Lys Glu Ile Asp Asp
Thr Val Thr Ser Asn Asn Asn Tyr Glu450 455
460Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala Pro465
470 475 480Gly Leu Ser Asp
Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala Tyr485 490
495Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln
His Asp500 505 510Val Asn Glu Leu Asn Val
Phe Phe Tyr Leu Asp Ala Gln Lys Val Pro515 520
525Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala
Leu530 535 540Leu Glu Gln Pro Lys Ile Tyr
Thr Phe Phe Ser Ser Glu Phe Ile Asn545 550
555 560Asn Val Asn Lys Pro Val Gln Ala Ala Leu Phe Val
Ser Trp Ile Gln565 570 575Gln Val Leu Val
Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr Val580 585
590Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly
Leu Ala595 600 605Leu Asn Ile Gly Asn Glu
Ala Gln Lys Gly Asn Phe Lys Asp Ala Leu610 615
620Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro Glu Leu
Leu625 630 635 640Ile Pro
Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser Ser645
650 655Asp Asn Lys Asn Lys Val Ile Lys Ala Ile Asn Asn
Ala Leu Lys Glu660 665 670Arg Asp Glu Lys
Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn Trp675 680
685Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln
Met Tyr690 695 700Gln Ala Leu Gln Asn Gln
Val Asn Ala Ile Lys Thr Ile Ile Glu Ser705 710
715 720Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn
Glu Leu Thr Asn Lys725 730 735Tyr Asp Ile
Lys Gln Ile Glu Asn Glu Leu Asn Gln Lys Val Ser Ile740
745 750Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser
Ser Ile Ser Tyr755 760 765Leu Met Lys Ile
Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu Tyr770 775
780Asp Glu Asn Val Lys Thr Tyr Leu Leu Asn Tyr Ile Ile Gln
His Gly785 790 795 800Ser
Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Thr Asp805
810 815Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser
Ser Tyr Thr Asp Asp820 825 830Lys Ile Leu
Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys Ser835
840 845Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys
Tyr Val Asp Thr850 855 860Ser Gly Tyr Asp
Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys Tyr865 870
875 880Pro Thr Asn Lys Asn Gln Phe Gly Ile
Tyr Asn Asp Lys Leu Ser Glu885 890 895Val
Asn Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr Lys900
905 910Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro
Asn Tyr Asp Asn Lys915 920 925Ile Val Asn
Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg Asp930
935 940Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn
Glu Ile Ile Trp945 950 955
960Thr Phe Glu Asp Asn Arg Gly Ile Asn Gln Lys Leu Ala Phe Asn Tyr965
970 975Gly Asn Ala Asn Gly Ile Ser Asp Tyr
Ile Asn Lys Trp Ile Phe Val980 985 990Thr
Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn Gly995
1000 1005Asn Leu Ile Asp Gln Lys Ser Ile Leu Asn Leu
Gly Asn Ile His Val1010 1015 1020Ser Asp
Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg Tyr1025
1030 1035 1040Ile Gly Ile Arg Tyr Phe Asn
Ile Phe Asp Lys Glu Leu Asp Glu Thr1045 1050
1055Glu Ile Gln Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn Ile Leu Lys1060
1065 1070Asp Phe Trp Gly Asn Tyr Leu Leu Tyr
Asp Lys Glu Tyr Tyr Leu Leu1075 1080
1085Asn Val Leu Lys Pro Asn Asn Phe Ile Asp Arg Arg Lys Asp Ser Thr1090
1095 1100Leu Ser Ile Asn Asn Ile Arg Ser Thr
Ile Leu Leu Ala Asn Arg Leu1105 1110 1115
1120Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser
Ser Thr1125 1130 1135Asn Asp Asn Leu Val
Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe Val1140 1145
1150Ala Ser Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr
Thr1155 1160 1165Asn Lys Glu Lys Thr Ile
Lys Ile Ser Ser Ser Gly Asn Arg Phe Asn1170 1175
1180Gln Val Val Val Met Asn Ser Val Gly Asn Cys Thr Met Asn Phe
Lys1185 1190 1195 1200Asn Asn
Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp Thr1205
1210 1215Val Val Ala Ser Thr Leu Phe Tyr Thr His Met Arg
Asp His Thr Asn1220 1225 1230Ser Asn Gly
Cys Phe Trp Asn Phe Ile Ser Glu Glu His Gly Trp Gln1235
1240 1245Glu Lys1250101065PRTClostridium botulinum 10Pro
Lys Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp Arg Thr1
5 10 15Ile Leu Tyr Ile Lys Pro Gly Gly
Ser Gln Glu Phe Tyr Lys Ser Phe20 25
30Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile Gly35
40 45Thr Thr Pro Gln Asp Phe His Pro Pro Thr
Ser Leu Lys Asn Gly Asp50 55 60Ser Ser
Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Glu Glu Lys Asp65
70 75 80Arg Phe Leu Lys Ile Val Thr
Lys Ile Phe Asn Arg Ile Asn Asn Asn85 90
95Leu Ser Gly Gly Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro Tyr100
105 110Leu Gly Asn Asp Asn Thr Pro Asp Asn
Gln Phe His Ile Gly Asp Ala115 120 125Ser
Ala Val Glu Ile Lys Phe Ser Asn Gly Ser Gln Asp Ile Leu Leu130
135 140Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp
Leu Phe Glu Thr Asn145 150 155
160Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His
Arg165 170 175Phe Gly Ser Ile Ala Ile Val
Thr Phe Ser Pro Glu Tyr Ser Phe Arg180 185
190Phe Asn Asp Asn Cys Met Asn Glu Phe Ile Gln Asp Pro Ala Leu Thr195
200 205Leu Met His Gln Leu Ile Tyr Ser Leu
His Gly Leu Tyr Gly Ala Lys210 215 220Gly
Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu Ile225
230 235 240Thr Asn Ile Arg Gly Thr
Asn Ile Glu Glu Phe Leu Thr Phe Gly Gly245 250
255Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr
Thr260 265 270Asn Leu Leu Ala Asp Tyr Lys
Lys Ile Ala Ser Lys Leu Ser Lys Val275 280
285Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu Ala290
295 300Lys Tyr Gly Leu Asp Lys Asp Ala Ser
Gly Ile Tyr Ser Val Asn Ile305 310 315
320Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr
Glu Phe325 330 335Asp Leu Arg Thr Lys Phe
Gln Val Lys Ser Arg Gln Thr Tyr Ile Gly340 345
350Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile
Tyr355 360 365Asn Ile Ser Glu Gly Tyr Asn
Ile Asn Asn Leu Lys Val Asn Phe Arg370 375
380Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr Gly385
390 395 400Arg Gly Leu Val
Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val Ser405 410
415Val Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn
Gly Glu420 425 430Leu Phe Phe Val Ala Ser
Glu Asn Ser Tyr Asn Asp Asp Asn Ile Asn435 440
445Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr
Glu450 455 460Asn Asp Leu Asp Gln Val Ile
Leu Asn Phe Asn Ser Glu Ser Ala Pro465 470
475 480Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln
Asn Asp Ala Tyr485 490 495Ile Pro Lys Tyr
Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln His Asp500 505
510Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln Lys
Val Pro515 520 525Glu Gly Glu Asn Asn Val
Asn Leu Thr Ser Ser Ile Asp Thr Ala Leu530 535
540Leu Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile
Asn545 550 555 560Asn Val
Asn Lys Pro Val Gln Ala Ala Leu Phe Val Ser Trp Ile Gln565
570 575Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln
Lys Ser Thr Val580 585 590Asp Lys Ile Ala
Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu Ala595 600
605Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp
Ala Leu610 615 620Glu Leu Leu Gly Ala Gly
Ile Leu Leu Glu Phe Glu Pro Glu Leu Leu625 630
635 640Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser
Phe Leu Gly Ser Ser645 650 655Asp Asn Lys
Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys Glu660
665 670Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile
Val Ser Asn Trp675 680 685Met Thr Lys Ile
Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met Tyr690 695
700Gln Ala Leu Gln Asn Gln Val Asn Ala Ile Lys Thr Ile Ile
Glu Ser705 710 715 720Lys
Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn Lys725
730 735Tyr Asp Ile Lys Gln Ile Glu Asn Glu Leu Asn
Gln Lys Val Ser Ile740 745 750Ala Met Asn
Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser Tyr755
760 765Leu Met Lys Ile Ile Asn Glu Val Lys Ile Asn Lys
Leu Arg Glu Tyr770 775 780Asp Glu Asn Val
Lys Thr Tyr Leu Leu Asn Tyr Ile Ile Gln His Gly785 790
795 800Ser Ile Leu Gly Glu Ser Gln Gln Glu
Leu Asn Ser Met Val Thr Asp805 810 815Thr
Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp Asp820
825 830Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe
Lys Arg Ile Lys Ser835 840 845Ser Ser Val
Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp Thr850
855 860Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp
Val Tyr Lys Tyr865 870 875
880Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser Glu885
890 895Val Asn Ile Ser Gln Asn Asp Tyr Ile
Ile Tyr Asp Asn Lys Tyr Lys900 905 910Asn
Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn Lys915
920 925Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile
Asn Cys Met Arg Asp930 935 940Asn Asn Ser
Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile Trp945
950 955 960Thr Phe Glu Asp Asn Arg Gly
Ile Asn Gln Lys Leu Ala Phe Asn Tyr965 970
975Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe Val980
985 990Thr Ile Thr Asn Asp Arg Leu Gly Asp
Ser Lys Leu Tyr Ile Asn Gly995 1000 1005Asn
Leu Ile Asp Gln Lys Ser Ile Leu Asn Leu Gly Asn Ile His Val1010
1015 1020Ser Asp Asn Ile Leu Phe Lys Ile Val Asn Cys
Ser Tyr Thr Arg Tyr1025 1030 1035
1040Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu
Thr1045 1050 1055Glu Ile Gln Thr Leu Tyr
Ser Asn Glu1060 106511844PRTClostridium butyricum 11Pro
Thr Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asn Arg Thr1
5 10 15Ile Leu Tyr Ile Lys Pro Gly Gly
Ser Gln Gln Phe Tyr Lys Ser Phe20 25
30Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile Gly35
40 45Thr Ile Pro Gln Asp Phe Leu Pro Pro Thr
Ser Leu Lys Asn Gly Asp50 55 60Ser Ser
Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Gln Glu Lys Asp65
70 75 80Lys Phe Leu Lys Ile Val Thr
Lys Ile Phe Asn Arg Ile Asn Asp Asn85 90
95Leu Ser Gly Arg Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro Tyr100
105 110Leu Gly Asn Asp Asn Thr Pro Asp Gly
Asp Phe Ile Ile Asn Asp Ala115 120 125Ser
Ala Val Pro Ile Gln Phe Ser Asn Gly Ser Gln Ser Ile Leu Leu130
135 140Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp
Leu Phe Glu Thr Asn145 150 155
160Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His
Gly165 170 175Phe Gly Ser Ile Ala Ile Val
Thr Phe Ser Pro Glu Tyr Ser Phe Arg180 185
190Phe Lys Asp Asn Ser Met Asn Glu Phe Ile Gln Asp Pro Ala Leu Thr195
200 205Leu Met His Glu Leu Ile His Ser Leu
His Gly Leu Tyr Gly Ala Lys210 215 220Gly
Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu Ile225
230 235 240Thr Asn Ile Arg Gly Thr
Asn Ile Glu Glu Phe Leu Thr Phe Gly Gly245 250
255Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr
Thr260 265 270Asn Leu Leu Ala Asp Tyr Lys
Lys Ile Ala Ser Lys Leu Ser Lys Val275 280
285Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu Ala290
295 300Lys Tyr Gly Leu Asp Lys Asp Ala Ser
Gly Ile Tyr Ser Val Asn Ile305 310 315
320Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr
Glu Phe325 330 335Asp Leu Ala Thr Lys Phe
Gln Val Lys Ser Arg Gln Thr Tyr Ile Gly340 345
350Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile
Tyr355 360 365Asn Ile Ser Glu Gly Tyr Asn
Ile Asn Asn Leu Lys Val Asn Phe Arg370 375
380Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr Gly385
390 395 400Arg Gly Leu Val
Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val Ser405 410
415Val Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn
Gly Glu420 425 430Leu Phe Phe Val Ala Ser
Glu Asn Ser Tyr Asn Asp Asp Asn Ile Asn435 440
445Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr
Glu450 455 460Asn Asp Leu Asp Gln Val Ile
Leu Asn Phe Asn Ser Glu Ser Ala Pro465 470
475 480Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln
Asn Asp Ala Tyr485 490 495Ile Pro Lys Tyr
Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln His Asp500 505
510Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln Lys
Val Pro515 520 525Glu Gly Glu Asn Asn Val
Asn Leu Thr Ser Ser Ile Asp Thr Ala Leu530 535
540Leu Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile
Asn545 550 555 560Asn Val
Asn Lys Pro Val Gln Ala Ala Leu Phe Val Gly Trp Ile Gln565
570 575Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln
Lys Ser Thr Val580 585 590Asp Lys Ile Ala
Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu Ala595 600
605Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp
Ala Leu610 615 620Glu Leu Leu Gly Ala Gly
Ile Leu Leu Glu Phe Glu Pro Glu Leu Leu625 630
635 640Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser
Phe Leu Gly Ser Ser645 650 655Asp Asn Lys
Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys Glu660
665 670Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile
Val Ser Asn Trp675 680 685Met Thr Lys Ile
Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met Tyr690 695
700Gln Ala Leu Gln Asn Gln Val Asn Ala Leu Lys Ala Ile Ile
Glu Ser705 710 715 720Lys
Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn Lys725
730 735Tyr Asp Ile Glu Gln Ile Glu Asn Glu Leu Asn
Gln Lys Val Ser Ile740 745 750Ala Met Asn
Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser Tyr755
760 765Leu Met Lys Leu Ile Asn Glu Val Lys Ile Asn Lys
Leu Arg Glu Tyr770 775 780Asp Glu Asn Val
Lys Thr Tyr Leu Leu Asp Tyr Ile Ile Lys His Gly785 790
795 800Ser Ile Leu Gly Glu Ser Gln Gln Glu
Leu Asn Ser Met Val Ile Asp805 810 815Thr
Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp Asp820
825 830Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe
Lys835 84012844PRTClostridium butyricum 12Pro Thr Ile Asn
Ser Phe Asn Tyr Asn Asp Pro Val Asn Asn Arg Thr1 5
10 15Ile Leu Tyr Ile Lys Pro Gly Gly Ser Gln Gln
Phe Tyr Lys Ser Phe20 25 30Asn Ile Met
Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile Gly35 40
45Thr Ile Pro Gln Asp Phe Leu Pro Pro Thr Ser Leu Lys
Asn Gly Asp50 55 60Ser Ser Tyr Tyr Asp
Pro Asn Tyr Leu Gln Ser Asp Gln Glu Lys Asp65 70
75 80Lys Phe Leu Lys Ile Val Thr Lys Ile Phe
Asn Arg Ile Asn Asp Asn85 90 95Leu Ser
Gly Arg Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro Tyr100
105 110Leu Gly Asn Asp Asn Thr Pro Asp Gly Asp Phe Ile
Ile Asn Asp Ala115 120 125Ser Ala Val Pro
Ile Gln Phe Ser Asn Gly Ser Gln Ser Ile Leu Leu130 135
140Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu
Thr Asn145 150 155 160Ser
Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His Gly165
170 175Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro
Glu Tyr Ser Phe Arg180 185 190Phe Lys Asp
Asn Ser Met Asn Glu Phe Ile Gln Asp Pro Ala Leu Thr195
200 205Leu Met His Gln Leu Ile Tyr Ser Leu His Gly Leu
Tyr Gly Ala Lys210 215 220Gly Ile Thr Thr
Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu Ile225 230
235 240Thr Asn Ile Arg Gly Thr Asn Ile Glu
Glu Phe Leu Thr Phe Gly Gly245 250 255Thr
Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr Thr260
265 270Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser
Lys Leu Ser Lys Val275 280 285Gln Val Ser
Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu Ala290
295 300Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr
Ser Val Asn Ile305 310 315
320Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu Phe325
330 335Asp Leu Ala Thr Lys Phe Gln Val Lys
Ser Arg Gln Thr Tyr Ile Gly340 345 350Gln
Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile Tyr355
360 365Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu
Lys Val Asn Phe Arg370 375 380Gly Gln Asn
Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr Gly385
390 395 400Arg Gly Leu Val Lys Lys Ile
Ile Arg Phe Cys Lys Asn Ile Val Ser405 410
415Val Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly Glu420
425 430Leu Phe Phe Val Ala Ser Glu Asn Ser
Tyr Asn Asp Asp Asn Ile Asn435 440 445Thr
Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr Glu450
455 460Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn
Ser Glu Ser Ala Pro465 470 475
480Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala
Tyr485 490 495Ile Pro Lys Tyr Asp Ser Asn
Gly Thr Ser Asp Ile Glu Gln His Asp500 505
510Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln Lys Val Pro515
520 525Glu Gly Glu Asn Asn Val Asn Leu Thr
Ser Ser Ile Asp Thr Ala Leu530 535 540Leu
Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile Asn545
550 555 560Asn Val Asn Lys Pro Val
Gln Ala Ala Leu Phe Val Gly Trp Ile Gln565 570
575Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr
Val580 585 590Asp Lys Ile Ala Asp Ile Ser
Ile Val Val Pro Tyr Ile Gly Leu Ala595 600
605Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala Leu610
615 620Glu Leu Leu Gly Ala Gly Ile Leu Leu
Glu Phe Glu Pro Glu Leu Leu625 630 635
640Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly
Ser Ser645 650 655Asp Asn Lys Asn Lys Val
Ile Lys Ala Ile Asn Asn Ala Leu Lys Glu660 665
670Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn
Trp675 680 685Met Thr Lys Ile Asn Thr Gln
Phe Asn Lys Arg Lys Glu Gln Met Tyr690 695
700Gln Ala Leu Gln Asn Gln Val Asn Ala Leu Lys Ala Ile Ile Glu Ser705
710 715 720Lys Tyr Asn Ser
Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn Lys725 730
735Tyr Asp Ile Glu Gln Ile Glu Asn Glu Leu Asn Gln Lys Val
Ser Ile740 745 750Ala Met Asn Asn Ile Asp
Arg Phe Leu Thr Glu Ser Ser Ile Ser Tyr755 760
765Leu Met Lys Leu Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu
Tyr770 775 780Asp Glu Asn Val Lys Thr Tyr
Leu Leu Asp Tyr Ile Ile Lys His Gly785 790
795 800Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser
Met Val Ile Asp805 810 815Thr Leu Asn Asn
Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp Asp820 825
830Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys835
840131250PRTClostridium butyricum 13Pro Thr Ile Asn Ser Phe Asn
Tyr Asn Asp Pro Val Asn Asn Arg Thr1 5 10
15Ile Leu Tyr Ile Lys Pro Gly Gly Ser Gln Gln Phe Tyr Lys
Ser Phe20 25 30Asn Ile Met Lys Asn Ile
Trp Ile Ile Pro Glu Arg Asn Val Ile Gly35 40
45Thr Ile Pro Gln Asp Phe Leu Pro Pro Thr Ser Leu Lys Asn Gly Asp50
55 60Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu
Gln Ser Asp Gln Glu Lys Asp65 70 75
80Lys Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn
Asp Asn85 90 95Leu Ser Gly Arg Ile Leu
Leu Glu Glu Leu Ser Lys Ala Asn Pro Tyr100 105
110Leu Gly Asn Asp Asn Thr Pro Asp Gly Asp Phe Ile Ile Asn Asp
Ala115 120 125Ser Ala Val Pro Ile Gln Phe
Ser Asn Gly Ser Gln Ser Ile Leu Leu130 135
140Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr Asn145
150 155 160Ser Ser Asn Ile
Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His Gly165 170
175Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser
Phe Arg180 185 190Phe Lys Asp Asn Ser Met
Asn Glu Phe Ile Gln Asp Pro Ala Leu Thr195 200
205Leu Met His Gln Leu Ile Tyr Ser Leu His Gly Leu Tyr Gly Ala
Lys210 215 220Gly Ile Thr Thr Lys Tyr Thr
Ile Thr Gln Lys Gln Asn Pro Leu Ile225 230
235 240Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu
Thr Phe Gly Gly245 250 255Thr Asp Leu Asn
Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr Thr260 265
270Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser
Lys Val275 280 285Gln Val Ser Asn Pro Leu
Leu Asn Pro Tyr Lys Asp Val Phe Glu Ala290 295
300Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn
Ile305 310 315 320Asn Lys
Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu Phe325
330 335Asp Leu Ala Thr Lys Phe Gln Val Lys Ser Arg Gln
Thr Tyr Ile Gly340 345 350Gln Tyr Lys Tyr
Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile Tyr355 360
365Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn
Phe Arg370 375 380Gly Gln Asn Ala Asn Leu
Asn Pro Arg Ile Ile Thr Pro Ile Thr Gly385 390
395 400Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys
Lys Asn Ile Val Ser405 410 415Val Lys Gly
Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly Glu420
425 430Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp
Asp Asn Ile Asn435 440 445Thr Pro Lys Glu
Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr Glu450 455
460Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser
Ala Pro465 470 475 480Gly
Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala Tyr485
490 495Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp
Ile Glu Gln His Asp500 505 510Val Asn Glu
Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln Lys Val Pro515
520 525Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile
Asp Thr Ala Leu530 535 540Leu Glu Gln Pro
Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile Asn545 550
555 560Asn Val Asn Lys Pro Val Gln Ala Ala
Leu Phe Val Gly Trp Ile Gln565 570 575Gln
Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr Val580
585 590Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro
Tyr Ile Gly Leu Ala595 600 605Leu Asn Ile
Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala Leu610
615 620Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu
Pro Glu Leu Leu625 630 635
640Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser Ser645
650 655Asp Asn Lys Asn Lys Val Ile Lys Ala
Ile Asn Asn Ala Leu Lys Glu660 665 670Arg
Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn Trp675
680 685Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg
Lys Glu Gln Met Tyr690 695 700Gln Ala Leu
Gln Asn Gln Val Asn Ala Leu Lys Ala Ile Ile Glu Ser705
710 715 720Lys Tyr Asn Ser Tyr Thr Leu
Glu Glu Lys Asn Glu Leu Thr Asn Lys725 730
735Tyr Asp Ile Glu Gln Ile Glu Asn Glu Leu Asn Gln Lys Val Ser Ile740
745 750Ala Met Asn Asn Ile Asp Arg Phe Leu
Thr Glu Ser Ser Ile Ser Tyr755 760 765Leu
Met Lys Leu Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu Tyr770
775 780Asp Glu Asn Val Lys Thr Tyr Leu Leu Asp Tyr
Ile Ile Lys His Gly785 790 795
800Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Ile
Asp805 810 815Thr Leu Asn Asn Ser Ile Pro
Phe Lys Leu Ser Ser Tyr Thr Asp Asp820 825
830Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys Ser835
840 845Ser Ser Val Leu Asn Met Arg Tyr Lys
Asn Asp Lys Tyr Val Asp Thr850 855 860Ser
Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys Tyr865
870 875 880Pro Thr Asn Lys Asn Gln
Phe Gly Ile Tyr Asn Asp Lys Leu Ser Glu885 890
895Val Asn Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr
Lys900 905 910Asn Phe Ser Ile Ser Phe Trp
Val Arg Ile Pro Asn Tyr Asp Asn Lys915 920
925Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg Asp930
935 940Asn Asn Ser Gly Trp Lys Val Ser Leu
Asn His Asn Glu Ile Ile Trp945 950 955
960Thr Leu Gln Asp Asn Ser Gly Ile Asn Gln Lys Leu Ala Phe
Asn Tyr965 970 975Gly Asn Ala Asn Gly Ile
Ser Asp Tyr Ile Asn Lys Trp Ile Phe Val980 985
990Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn
Gly995 1000 1005Asn Leu Ile Asp Lys Lys Ser
Ile Leu Asn Leu Gly Asn Ile His Val1010 1015
1020Ser Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg Tyr1025
1030 1035 1040Ile Gly Ile Arg
Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu Thr1045 1050
1055Glu Ile Gln Thr Leu Tyr Asn Asn Glu Pro Asn Ala Asn Ile
Leu Lys1060 1065 1070Asp Phe Trp Gly Asn
Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu Leu1075 1080
1085Asn Val Leu Lys Pro Asn Asn Phe Ile Asn Arg Arg Thr Asp Ser
Thr1090 1095 1100Leu Ser Ile Asn Asn Ile
Arg Ser Thr Ile Leu Leu Ala Asn Arg Leu1105 1110
1115 1120Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val
Asn Asn Ser Ser Thr1125 1130 1135Asn Asp
Asn Leu Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe Val1140
1145 1150Ala Ser Lys Thr His Leu Leu Pro Leu Tyr Ala Asp
Thr Ala Thr Thr1155 1160 1165Asn Lys Glu
Lys Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe Asn1170
1175 1180Gln Val Val Val Met Asn Ser Val Gly Asn Cys Thr
Met Asn Phe Lys1185 1190 1195
1200Asn Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp Thr1205
1210 1215Val Val Ala Ser Thr Leu Phe Tyr Thr
His Met Arg Asp Asn Thr Asn1220 1225
1230Ser Asn Gly Phe Phe Trp Asn Phe Ile Ser Glu Glu His Gly Trp Gln1235
1240 1245Glu Lys1250141065PRTClostridium
butyricum 14Pro Lys Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp Arg
Thr1 5 10 15Ile Leu Tyr
Ile Lys Pro Gly Gly Ser Gln Glu Phe Tyr Lys Ser Phe20 25
30Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn
Val Ile Gly35 40 45Thr Thr Pro Gln Asp
Phe His Pro Pro Thr Ser Leu Lys Asn Gly Asp50 55
60Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Glu Glu Lys
Asp65 70 75 80Arg Phe
Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asn Asn85
90 95Leu Ser Gly Gly Ile Leu Leu Glu Glu Leu Ser Lys
Ala Asn Pro Tyr100 105 110Leu Gly Asn Asp
Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp Ala115 120
125Ser Ala Val Glu Ile Lys Phe Ser Asn Gly Ser Gln Asp Ile
Leu Leu130 135 140Pro Asn Val Ile Ile Met
Gly Ala Glu Pro Asp Leu Phe Glu Thr Asn145 150
155 160Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met
Pro Ser Asn His Arg165 170 175Phe Gly Ser
Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe Arg180
185 190Phe Asn Asp Asn Cys Met Asn Glu Phe Ile Gln Asp
Pro Ala Leu Thr195 200 205Leu Met His Gln
Leu Ile Tyr Ser Leu His Gly Leu Tyr Gly Ala Lys210 215
220Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro
Leu Ile225 230 235 240Thr
Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly Gly245
250 255Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser
Asn Asp Ile Tyr Thr260 265 270Asn Leu Leu
Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys Val275
280 285Gln Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp
Val Phe Glu Ala290 295 300Lys Tyr Gly Leu
Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn Ile305 310
315 320Asn Lys Phe Asn Asp Ile Phe Lys Lys
Leu Tyr Ser Phe Thr Glu Phe325 330 335Asp
Leu Arg Thr Lys Phe Gln Val Lys Ser Arg Gln Thr Tyr Ile Gly340
345 350Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu
Asn Asp Ser Ile Tyr355 360 365Asn Ile Ser
Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe Arg370
375 380Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr
Pro Ile Thr Gly385 390 395
400Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val Ser405
410 415Val Lys Gly Ile Arg Lys Ser Ile Cys
Ile Glu Ile Asn Asn Gly Glu420 425 430Leu
Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile Asn435
440 445Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser
Asn Asn Asn Tyr Glu450 455 460Asn Asp Leu
Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala Pro465
470 475 480Gly Leu Ser Asp Glu Lys Leu
Asn Leu Thr Ile Gln Asn Asp Ala Tyr485 490
495Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln His Asp500
505 510Val Asn Glu Leu Asn Val Phe Phe Tyr
Leu Asp Ala Gln Lys Val Pro515 520 525Glu
Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala Leu530
535 540Leu Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser
Ser Glu Phe Ile Asn545 550 555
560Asn Val Asn Lys Pro Val Gln Ala Ala Leu Phe Val Ser Trp Ile
Gln565 570 575Gln Val Leu Val Asp Phe Thr
Thr Glu Ala Asn Gln Lys Ser Thr Val580 585
590Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu Ala595
600 605Leu Asn Ile Gly Asn Glu Ala Gln Lys
Gly Asn Phe Lys Asp Ala Leu610 615 620Glu
Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro Glu Leu Leu625
630 635 640Ile Pro Thr Ile Leu Val
Phe Thr Ile Lys Ser Phe Leu Gly Ser Ser645 650
655Asp Asn Lys Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys
Glu660 665 670Arg Asp Glu Lys Trp Lys Glu
Val Tyr Ser Phe Ile Val Ser Asn Trp675 680
685Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met Tyr690
695 700Gln Ala Leu Gln Asn Gln Val Asn Ala
Ile Lys Thr Ile Ile Glu Ser705 710 715
720Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr
Asn Lys725 730 735Tyr Asp Ile Lys Gln Ile
Glu Asn Glu Leu Asn Gln Lys Val Ser Ile740 745
750Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser
Tyr755 760 765Leu Met Lys Ile Ile Asn Glu
Val Lys Ile Asn Lys Leu Arg Glu Tyr770 775
780Asp Glu Asn Val Lys Thr Tyr Leu Leu Asn Tyr Ile Ile Gln His Gly785
790 795 800Ser Ile Leu Gly
Glu Ser Gln Gln Glu Leu Asn Ser Met Val Thr Asp805 810
815Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr
Asp Asp820 825 830Lys Ile Leu Ile Ser Tyr
Phe Asn Lys Phe Phe Lys Arg Ile Lys Ser835 840
845Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp
Thr850 855 860Ser Gly Tyr Asp Ser Asn Ile
Asn Ile Asn Gly Asp Val Tyr Lys Tyr865 870
875 880Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp
Lys Leu Ser Glu885 890 895Val Asn Ile Ser
Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr Lys900 905
910Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp
Asn Lys915 920 925Ile Val Asn Val Asn Asn
Glu Tyr Thr Ile Ile Asn Cys Met Arg Asp930 935
940Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile
Trp945 950 955 960Thr Phe
Glu Asp Asn Arg Gly Ile Asn Gln Lys Leu Ala Phe Asn Tyr965
970 975Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys
Trp Ile Phe Val980 985 990Thr Ile Thr Asn
Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn Gly995 1000
1005Asn Leu Ile Asp Gln Lys Ser Ile Leu Asn Leu Gly Asn Ile
His Val1010 1015 1020Ser Asp Asn Ile Leu
Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg Tyr1025 1030
1035 1040Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp
Lys Glu Leu Asp Glu Thr1045 1050 1055Glu
Ile Gln Thr Leu Tyr Ser Asn Glu1060 1065
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