Mutants of porcine trypsin

Abstract

To provide polypeptide variants of porcine trypsin, nucleic acid molecules encoding these variants, and host cells comprising such nucleic acid molecules.SOLUTION: A variant of porcine trypsin comprises or consists of an amino acid sequence that has at least 90% sequence identity to native porcine trypsin represented by a specific sequence, where the amino acid sequence contains at least one or more amino acid substitutions at one or more positions corresponding to F24, S44, D56, G78, Y131, S172 and W193 of native porcine trypsin represented by the specific sequence, with the proviso that the amino acid sequence is not native porcine trypsin according to the specific sequence, and with the proviso that the amino acid sequence is not porcine variant trypsin S172A according to another specific sequence.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to peptide variants of porcine trypsin, nucleic acid molecules encoding these variants, and host cells containing such nucleic acid molecules. The present invention also relates to the use of these variants in a method for producing insulin. The present invention further relates to the use of these variants as a medicament, as a food ingredient, or as a feed ingredient, and to the use of these variants in a method for producing a food ingredient or a feed ingredient.

Background Art

[0002] The endopeptidase trypsin is used for the production of human insulin, insulin analogs, and also insulin derivatives. Human insulin, insulin analogs, or insulin derivatives are produced from preproinsulin (PPI) by enzymatic hydrolysis using trypsin, in which the presequence and C-peptide are cleaved to obtain the respective products (see FIG. 1). Trypsin is specific for cleavage on the C-terminal side of Arg and Lys residues within the polypeptide chain. In preprohuman insulin and preproinsulin glargine polypeptides, there are several Arg and Lys residues, including B29Lys, B31Arg, and B32Arg at the junction of the B-chain and C-peptide. Therefore, preprohuman insulin and preproinsulin glargine are cleaved by trypsin on the C-terminal side of Arg and Lys residues, including the C-terminal side of B29Lys, B31Arg, and B32Arg. In the case of the production of insulin glargine, trypsin cleavage after B32Arg provides the final insulin glargine. Trypsin cleavage after B29Lys and B31Arg results in the by-products desB30-Thr ("des-Thr")-insulin glargine and desB32Arg-insulin glargine (also called "des-Arg"). Therefore, it is necessary to remove by-products in the method for producing insulin glargine.

[0003] In the production of human insulin, trypsin cleavage after B31Arg results in B31Arg-human insulin (also called "mono-Arg"), and trypsin cleavage after B32Arg results in B31Arg-B32Arg-human insulin (also called "di-Arg"). Both are intermediates in the production method. In subsequent production methods, both of these intermediates are converted to the final human insulin. Trypsin cleavage after B29Lys results in the by-product desB30-Thr ("des-Thr")-human insulin. Therefore, it is necessary to remove by-products in the production method of human insulin.

[0004] In the production of human insulin and insulin glargine, trypsin cleavage after B32Arg results in the final insulin glargine. In the case of human insulin, trypsin cleavage after B32Arg results in an intermediate, which is converted to the final human insulin in subsequent process steps.

[0005] To address the unwanted formation of desB30-Thr-intermediates, past development has led to the porcine trypsin mutant S172A, which reduces this mis-cleavage due to its high selectivity for the C-terminal side of Arg over Lys. The porcine trypsin mutant S172A is described in Patent Document 1, which is hereby incorporated by reference in its entirety.

[0006] Here, the inventors carefully observed the kinetics of the trypsin S172A-catalyzed reaction and surprisingly found that the formation of mis-cleaved by-products is not only due to the main mis-cleavage of PPI by trypsin acting as an endo protease as described in the literature, but also due to the side-activity inherent in porcine trypsin and its mutant S172A acting as exopeptidases. Exopeptidase activity results in the degradation of insulin glargine under the formation of des-Arg and des-Thr.

Prior Art Documents

Patent Document

[0007]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] Technical problems underlying the present invention Therefore, in the prior art, there has been a need for novel trypsin variants with reduced side activity, particularly reduced exopeptidase activity. Such novel trypsin variants would reduce the amount of by-products in the cleavage of PPI and would result in improved yields of insulin (or insulin derivatives) when producing insulin (or insulin derivatives) from PPI.

[0009] The present inventors have produced novel variants of porcine trypsin having reduced exopeptidase activity and / or showing reduced formation of by-products in the PPI cleavage reaction.

[0010] The above summary does not necessarily explain all the problems solved by the present invention.

Means for Solving the Problems

[0011] In a first aspect, the present invention provides a variant of porcine trypsin, comprising or consisting of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, wherein the amino acid sequence differs from SEQ ID NO: 1 in that there is at least one or more amino acid substitutions at one or more positions corresponding to F24, S44, D56, G78, Y131, S172 and W193 of native porcine trypsin according to SEQ ID NO: 1, provided that The amino acid sequence is not the native porcine trypsin according to SEQ ID NO: 1; and The amino acid sequence is not the porcine mutant trypsin S172A according to SEQ ID NO: 2, a mutant of said porcine trypsin, relates to.

[0012] In a second aspect, the present invention relates to a nucleic acid molecule encoding the mutant porcine trypsin described in the first aspect.

[0013] In a third aspect, the present invention relates to a host cell containing the nucleic acid molecule described in the second aspect.

[0014] In a fourth aspect, the present invention is a method for producing the mutant porcine trypsin described in the first aspect, comprising: a step of culturing the host cell described in the third aspect, and a step of isolating the mutant of porcine trypsin from the culture medium or from the host cell relates to said method comprising.

[0015] In a fifth aspect, the present invention relates to the mutant porcine trypsin described in the first aspect, or the nucleic acid molecule described in the second aspect, or the host cell described in the third aspect for use as a medicament, for use as a food ingredient, for use as a feed ingredient, or for use in a method for producing a food ingredient or a feed ingredient.

[0016] In a sixth aspect, the present invention relates to the use of the mutant porcine trypsin described in the first aspect in a method for producing human insulin, an insulin analog or a derivative of insulin.

[0017] In a seventh aspect, the present invention is the use of the mutant porcine trypsin described in the first aspect for cleaving a protein or peptide having the general formula A-Lys-Thr-Arg-Arg-B, where A is an amino acid sequence consisting of one or more amino acids; and Regarding the above use, B is an amino acid sequence consisting of one or more amino acids.

[0018] In an eighth aspect, the present invention relates to a composition comprising a variant of porcine trypsin according to the first aspect, a nucleic acid molecule according to the second aspect, or a host cell according to the third aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0020] Definitions The present invention will be described in detail below, but it should be understood that the present invention is not limited to the specific methodologies, pro tocols and reagents described herein, and that they are subject to change. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0021] In the practice of the present invention, conventional techniques in chemistry, biochemistry, molecular biology, immunology, and recombinant DNA, as described in the literature of the art, are used unless otherwise indicated.

[0022] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to be interpreted as including the stated member, integer or step or group of members, integers or steps but not as excluding any other member, integer or step or group of members, integers or steps, provided that in some embodiments such other member, integer or step or group of members, integers or steps may be excluded, that is, the subject matter of the invention is intended to include the stated member, integer or step or group of members, integers or steps. The terms "a", "an" and "the" and similar references used in the context of describing the invention (in particular, in the context of the claims) are to be construed in this specification, unless the context clearly dictates otherwise or is inconsistent therewith, as covering both the singular and the plural. The recitation of a range of values herein is merely intended to serve as a shorthand way of referring individually to each separate value falling within the range. Unless otherwise indicated, each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless the context clearly dictates otherwise or is inconsistent therewith. The use of any and all examples or exemplary language (e.g., "such as") provided herein is merely intended to better illustrate the invention and does not impose a limitation on the scope of the invention unless a claim is made. Recitation in this specification of any aspect is not to be construed as indicating that any element is essential to the practice of the invention that is not recited in a claim.

[0023] Throughout the body of this specification, several documents (e.g., patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank Accession Number sequence registrations, etc.) are cited. Nothing in this specification should be construed as admitting that the present invention does not predate such disclosures on the grounds of prior invention. Some of the documents cited herein are characterized as being "incorporated by reference" or "incorporated by reference in their entirety." In the event of a conflict between the definitions or teachings of such incorporated documents and the definitions or teachings set forth herein, the text of this specification shall prevail.

[0024] Sequences: All sequences referred to in this specification are disclosed in the accompanying Sequence Listing, which forms part of this specification along with its entire content and disclosure.

[0025] When used in connection with biological materials such as nucleic acid molecules, (poly)peptides, host cells, etc., the term "natural" refers to materials found in nature and not manipulated by humans.

[0026] As used herein, the terms "amino acid" or "amino acid residue" each refer to natural amino acids, unnatural amino acids, amino acid analogs, and amino acid mimetics that function in a manner similar to natural amino acids, in their D and L stereoisomeric forms when their structure permits such stereoisomeric forms. Amino acids are referred to herein by either their names, their commonly known three-letter symbols, or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0027] ​When used in connection with amino acids, the term "natural" refers to the conventional 20 amino acids (i.e., alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y)), as well as selenocysteine, pyrrolysine (PYL), and pyrroline-carboxylic acid (PCL). The terms "natural amino acid" and "codable amino acid" are used interchangeably herein.

[0028] As used herein, the term "non-natural amino acid" means an amino acid that is not naturally encoded or not found in the genetic code of any organism. As used herein, the term "amino acid analog" refers to a compound having the same basic chemical structure as a natural amino acid. As used herein, the term "amino acid mimetic" refers to a compound having a structure that is different from the general chemical structure of an amino acid but functions in a manner similar to a natural amino acid.

[0029] As used herein, the term "peptide" refers to a polymeric form of amino acids of any length, for example, containing 2 or more, or 3 or more, or 4 or more, or 6 or more, or 8 or more, or 9 or more, or 10 or more, or 13 or more, or 16 or more, or 21 or more amino acids, covalently linked by peptide bonds. The term "polypeptide" refers to a large peptide, particularly a peptide having more than 100 amino acid residues. The terms "polypeptide" and "protein" are used interchangeably herein. This application provides amino acid sequence information for some of the peptides and polypeptides disclosed herein. In this specification, the amino acid sequence is presented from the amino terminus (left side) to the carboxy terminus (right side) of the peptide or polypeptide.

[0030] As used herein, a "variant" of a (poly)peptide refers to a (poly)peptide that differs from the corresponding reference (poly)peptide in that it has one or more amino acid modification substitutions in its amino acid sequence. In one embodiment, the "variant" maintains one or more biological activities of the reference (poly)peptide. The reference (poly)peptide can be a wild-type (poly)peptide or a non-natural (poly)peptide. The terms "variant" and "mutant" are used interchangeably herein.

[0031] As used herein, the term "variant of porcine trypsin" refers to a polypeptide that differs from the wild-type porcine trypsin shown in SEQ ID NO: 1 in that it has one or more amino acid modification substitutions in its amino acid sequence. The term "variant of porcine trypsin" particularly refers to a polypeptide that differs from the wild-type porcine trypsin shown in SEQ ID NO: 1 in that it has one or more amino acid exchange substitutions. According to the present invention, the "variant of porcine trypsin" exhibits an endoprotease activity similar to that of the reference trypsin (e.g., wild-type porcine trypsin according to SEQ ID NO: 1 or trypsin variant S172A according to SEQ ID NO: 2), but the endo... The endopeptidase activities can be different in terms of their selectivity for certain cleavage sites.

[0032] The term "amino acid modification" includes amino acid exchange, amino acid deletion, and amino acid addition. The terms "amino acid exchange" and "amino acid substitution" are used interchangeably herein. The term "amino acid deletion" includes N-terminal cleavage, internal deletion, and C-terminal cleavage. The term "amino acid addition" includes N-terminal addition, amino acid insertion, and C-terminal addition.

[0033] The expression "Xaa24" (and similar expressions such as Xaa44, Xaa56, Xaa78, etc.) refers to the variable amino acid at position 24 (or 44, 56, 78, etc., respectively) of a given amino acid sequence. The sequence listing supplied with this application contains a protein sequence, namely SEQ ID NO: 3, along with the positions of the variable amino acids. The positions of the variable amino acids within the protein sequence according to SEQ ID NO: 3 are indicated by the amino acid Xaa (in three-letter code format). In this example, the number 24 indicates that this variable position is the 24th amino acid of the protein sequence according to SEQ ID NO: 3. Similarly, the expression Xaa44 indicates that the 44th position of SEQ ID NO: 3 is the position of the variable amino acid, and so on.

[0034] As used herein, the term "human insulin" refers to the human hormone whose structure and properties are well known. Human insulin has two peptide chains (chain A and chain B), and the two peptide chains are linked by cysteine residues, i.e., by disulfide bridges between chain A and chain B. Chain A is a peptide of 21 amino acids, and chain B is a peptide of 30 amino acids, and the two chains are linked by three disulfide bridges: the first between the cysteines at positions 6 and 11 of chain A; the second between the cysteine at position 7 of chain A and the cysteine at position 7 of chain B; and the third between the cysteine at position 20 of chain A and the cysteine at position 19 of chain B.

[0035] As used herein, the terms "derivative of insulin" and "insulin derivative" refer to a peptide having a molecular structure that can formally be derived from the structure of native insulin, for example, the structure of human insulin, in which case one or more organic substituents (such as fatty acids) are attached to one or more amino acids. Optionally, one or more amino acids present in native insulin are deleted and / or replaced with other amino acids, including non-codable amino acids, or amino acids, including non-codable ones, are added to native insulin. Examples of insulin derivatives include, but are not limited to, the following: (i). "Insulin detemir", which differs from human insulin in that the C-terminal threonine at position B30 is removed and a fatty acid residue (myristic acid residue) is attached to the epsilon-amino functional group of lysine at position B29. (ii). "Insulin degludec", which differs from human insulin in that the last amino acid is deleted from the B chain and a glutamyl link from BLys29 to hexadecanedioic acid is added.

[0036] As used herein, the terms "analog of insulin" and "insulin analog" refer to a peptide having a molecular structure that can formally be derived from the structure of native insulin, for example, the structure of human insulin, by deleting and / or exchanging at least one amino acid residue present in native insulin and / or by adding at least one amino acid residue. The added and / or exchanged amino acid residues may be codable amino acid residues, other natural residues, or entirely synthetic amino acid residues. Examples of insulin ana logs include, but are not limited to, the following: (i). Insulin aspart is created by recombinant DNA technology to replace the amino acid B28 in human insulin, which is proline (i.e., the 28th amino acid in the B chain of human insulin), with aspartic acid; (ii). Insulin lispro is created by recombinant DNA technology such that the penultimate lysine residue and proline residue at the C-terminus of the B chain of human insulin are reversed (human insulin: B28Pro - B29Lys; insulin lispro: B28Lys - B29Pro); (iii). Insulin glulisine differs from human insulin in that the amino acid asparagine at position B3 is replaced with lysine and the lysine at position B29 is replaced with glutamic acid; (iv). Insulin glargine differs from human insulin in that the asparagine at position A21 is replaced with glycine and the B chain is extended at the carboxy terminus by two arginines.

[0037] As used herein, the term "yield" refers to the ratio of the resulting product obtained after trypsin cleavage (i.e., insulin or an insulin derivative or an insulin analog) to preproinsulin (or a preproinsulin derivative or a preproinsulin analog) before trypsin cleavage. The yield is influenced by the selectivity of the trypsin variant that cleaves at the desired cleavage site and by its exonuclease activity. The higher the selectivity of the trypsin variant that cleaves only at the desired cleavage site and the lower its exonuclease activity, the higher the yield.

[0038] For example, the "yield of insulin glargine" or the "yield of B31Arg-B32Arg-human insulin" refers to the ratio of the resulting insulin glargine or B31Arg-B32Arg-human insulin obtained after trypsin cleavage to the respective preproinsulin glargine or human preproinsulin before trypsin cleavage. The "yield of insulin glargine" and the "yield of B31Arg-B32Arg-human insulin" are determined by the selectivity of the trypsin mutant that cleaves only after B32Arg and by its exonuclease activity. The higher the selectivity of the trypsin mutant that cleaves only after B32Arg and the lower its exonuclease activity, the higher the yield.

[0039] As used herein, the term "improved selectivity" refers to the property of a trypsin mutant that cleaves to a greater extent at a desired cleavage site and to a lesser extent at an undesired cleavage site compared to a reference trypsin. Improved selectivity will result in reduced formation of unwanted by-products. The reference trypsin can be, for example, wild-type porcine trypsin according to SEQ ID NO: 1 or trypsin mutant S172A according to SEQ ID NO: 2.

[0040] "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. Optimal alignment of sequences for comparison can be performed, in addition to by hand, by the local homology algorithm of Smith and Waterman, 1981, Adv. App. Math. 2, pages 482-489 (incorporated herein by reference), by the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, pages 443-453 (incorporated herein by reference), by the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, pages 2444-2448 (incorporated herein by reference), or by computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLASTP, BLASTN, and TFASTA of the Wisconsin Genetics Software Package, Gene tics Computer Group, 575 Science Drive, Madison, Wis.).

[0041] In the context of the present invention, unless otherwise specified, the degree of sequence identity between a modified sequence and the sequence from which it is derived is generally calculated with respect to the full length of the unmodified sequence. In embodiments where neither sequence is a modified sequence, unless otherwise specified, the degree of sequence identity is calculated with respect to the full length of the sequence defined as the reference sequence. In embodiments where neither sequence can be regarded as a reference sequence, unless otherwise specified, the degree of sequence identity is calculated with respect to the longer of the two sequences.

[0042] The "nucleic acid molecule" according to some embodiments of the present invention is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The nucleic acid molecule according to the present invention can be in the form of a single-stranded or double-stranded molecule; and can be linear or can form a ring by covalent closure.

[0043] The term "DNA" relates to molecules containing deoxyribonucleotide residues and molecules consisting entirely or substantially of deoxyribonucleotide residues. "Deoxyribonucleotide" relates to nucleotides lacking a hydroxyl group at the 2'-position of the beta-D-ribofuranosyl group. The term "DNA" includes isolated DNA such as partially or completely purified DNA, essentially pure DNA, synthetic DNA, and DNA produced by genetic recombination, and also includes modified DNA that differs from natural DNA in that it has one or more nucleotide additions, deletions, substitutions, and / or modified substitutions. Such modifications include the addition of non-nucleotide material at or within the ends of the DNA, for example, at one or more nucleotides of the DNA. The nucleotides in a DNA molecule can also include non-standard nucleotides such as, for example, unnatural nucleotides or chemically synthesized nucleotides. These modified DNAs can be referred to as analogs or analogs of natural DNA. When used in connection with nucleotides, the term "natural" refers to each of the bases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U).

[0044] The term "RNA" relates to molecules containing ribonucleotide residues and molecules consisting entirely or substantially of ribonucleotide residues. "Ribonucleotide" relates to a nucleotide having a hydroxyl group at the 2'-position of a beta-D-ribofuranosyl group. The term "RNA" includes isolated RNA such as partially or fully purified RNA, substantially pure RNA, synthetic RNA, and RNA produced by genetic recombination, and also includes modified RNA that differs from natural RNA in that it has one or more nucleotide additions, deletions, substitutions, and / or modified substitutions. Such modifications include the addition of non-nucleotide material at or within one or more nucleotides of the RNA, such as to the ends or interior of the RNA. The nucleotides in an RNA molecule can also include non-natural nucleotides or non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be referred to as analogs or analogs of natural RNA. According to the present invention, "RNA" refers to single-stranded RNA or double-stranded RNA. In one embodiment, the RNA is mRNA, for example, in vitro transcribed RNA (IVT RNA) or synthetic RNA. The RNA can also be modified, for example, by one or more modifications that improve the stability (e.g., half-life) of the RNA. Such modifications are known to those skilled in the art and include, for example, a 5'-cap or 5'-cap analog.

[0045] The nucleic acid molecule according to the present invention can be contained in / included in a vector. The term "vector" as used herein includes plasmid vectors, cosmid vectors All vectors known to those skilled in the art are included, including phage vectors such as lambda phage, viral vectors such as adenovirus vectors or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). The vectors include expression vectors as well as cloning vectors. Expression vectors include plasmids as well as viral vectors and generally contain the desired coding sequence necessary for the expression of an operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system, along with appropriate DNA sequences. Cloning vectors are generally used to manipulate and amplify a particular desired DNA fragment and may lack the functional sequences necessary for the expression of the desired DNA fragment.

[0046] Alternatively, the nucleic acid molecule according to the present invention may be integrated into the genome, for example, the genome of a host cell. Means and methods for integrating a particular nucleic acid molecule into the genome are known to those skilled in the art.

[0047] The terms "cell" or "host cell" generally refer to intact cells, i.e., cells with an intact membrane that have not released their normal intracellular components such as enzymes, organelles, or genetic material. Intact cells are generally viable cells, i.e., living cells that can perform their normal metabolic functions. As used herein, the terms refer to any cell that has been transfected or transformed with exogenous nucleic acid. In some embodiments, when transfected or transformed with exogenous nucleic acid and introduced into a recipient, the cell can express the nucleic acid in the recipient. The term "cell" includes prokaryotic cells such as bacterial cells, and eukaryotic cells such as yeast cells, fungal cells, or mammalian cells. Bacterial cells include Gram-negative bacterial strains such as strains of Escherichia coli, Proteus, and Pseudomonas, as well as Gram-positive bacterial strains such as strains of Bacillus, Streptomyces, Staphylococcus, and Lactococcus. Fungal cells include cells from species of Trichoderma, Neurospor, and Aspergillus. Yeast cells include species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica). Cells from species of the genus Pichia (e.g., Pichia methanolica), the genus Komagataella (e.g., Komagataella pastoris and Komagataella phaffii), and the genus Hansenula are included. Mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, HEK293, etc. Amphibian cells, insect cells, plant cells, and any other cells used in the art for the expression of heterologous proteins can also be used. Mammalian cells, such as cells from humans, mice, hamsters, pigs, goats, and primates, are particularly suitable for adoptive transfer. These cells can be derived from many tissue types, including cell lines such as primary cells and cells of the immune system, particularly antigen-presenting cells such as dendritic cells and T cells, stem cells such as hematopoietic stem cells and mesenchymal stem cells, and other cell types. An antigen-presenting cell is a cell that presents an antigen in the context of the major histocompatibility complex on its surface. T cells can recognize this complex using their T cell receptor (TCR). A "cell" or "host cell" is either isolated or part of a tissue or organism, particularly a " non-human organism".

[0048] As used herein, the term "non-human organism" means non-human primates or other animals, particularly mammals such as cows, horses, pigs, sheep, goats, dogs, cats, rabbits, or rodents, e.g., mice, rats, guinea pigs, and hamsters.

[0049] As used herein, the term "pharmaceutical" refers to a substance / composition used in a therapeutic method, i.e., in the treatment of diseases and disorders.

[0050] According to the present invention, the term "disease or disorder" refers to any pathological or unhealthy condition.

[0051] "To treat" means to administer to a subject a compound, composition, or combination of compounds or compositions for the purpose of preventing or eliminating a disease or disorder; for the purpose of arresting or attenuating a disease or disorder of interest; for the purpose of inhibiting or attenuating the development of a new disease or disorder in a subject; for the purpose of reducing the frequency or severity and / or recurrence of symptoms in a subject having or having had a disease or disorder; and / or for the purpose of extending, i.e., lengthening, the lifespan of a subject.

[0052] In particular, the term "treating a disease or disorder / treatment" includes curing, shortening the duration, improving, preventing, decelerating or inhibiting progression or worsening, or preventing or delaying the onset of a disease or disorder or its symptoms.

[0053] As used herein, the term "subject" means a subject for treatment, particularly an affected subject (also referred to as a "patient"), and includes humans, non-human primates, or other animals, particularly mammals such as cows, horses, pigs, sheep, goats, dogs, cats, rabbits, or rodents such as mice, rats, guinea pigs, and hamsters. In one embodiment, the subject / patient is a human.

[0054] A "composition" according to the present invention contains an effective amount of a variant of porcine trypsin of the present invention (also referred to herein as an "active agent"). The compositions according to the present invention can be used (a) in a production process, particularly in a production process for producing a peptide, particularly in a production process for producing human insulin, insulin derivatives and / or insulin analogs; (b) as a food ingredient; (c) as a feed ingredient; (d) or in a process for producing a food ingredient or a feed ingredient. The compositions according to the present invention can also be used for treating a disease or disorder and thus can be made into a pharmaceutical composition.

[0055] The "pharmaceutical composition" according to the present invention contains a therapeutically effective amount of a variant of porcine trypsin, a nucleic acid molecule, or a host cell (also referred to herein as an "active agent") of the present invention in order to produce a desired reaction or a desired effect. The pharmaceutical composition according to the present invention can further contain at least one other active agent. In one embodiment, the composition is provided in a uniform dosage form and can be manufactured by methods known per se. The composition can be in the form of, for example, a solution or a suspension.

[0056] The composition or the pharmaceutical composition can further contain one or more excipients. In one embodiment regarding the pharmaceutical composition, all of the one or more excipients are pharmaceutically acceptable. As used herein, "pharmaceutically acceptable" refers to being physiologically well-tolerated by mammals or humans. In particular, this means that it has been approved by a federal or state government regulatory agency for use in animals and more specifically, in humans, or is listed in the United States Pharmacopeia or other generally recognized pharmacopeias.

[0057] The composition or the pharmaceutical composition according to the present invention may exist in the form of a composition in which various active agents and excipients (e.g., diluents and / or carriers) are mixed with each other, or may take the form of a formulated preparation in which the active agents are present in a partially or completely different form. An example of such a combination or formulated preparation is a kit-of-parts.

[0058] Unless the context clearly indicates otherwise, the terms "active agent" and "active ingredient" refer to the variant of porcine trypsin of the present invention, the nucleic acid molecule of the present invention, and the host cell of the present invention. The terms "active agent" and "active ingredient" are used interchangeably herein.

[0059] The term "carrier" refers to a natural or synthetic organic or inorganic component with which an active agent is combined to facilitate, enhance, or enable application. According to the present invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents, adjuvants, excipients, vehicles, or encapsulating substances. Such carriers include, for example, saline and oils, including those of petroleum origin, animal origin, plant origin, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., and can be sterilized liquids. Saline as well as aqueous dextrose and glycerol solutions can also be used as liquid carriers. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. The composition or pharmaceutical composition can optionally also contain trace amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. The composition or pharmaceutical composition can be formulated as a suppository using traditional binders and carriers such as triglycerides. The compounds of the present invention can be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed with free amino groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with free carboxyl groups, such as those derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Such compositions will contain an effective amount of the compound, together with a suitable amount of carrier, and in some embodiments in a purified form. The formulation must be appropriate for the mode of administration.

[0060] As used herein, the term "excipient" is intended to include all substances that are not active agents but may be present in a composition or a pharmaceutical composition, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), carriers, lubricants, thickeners, surfactants, preservatives (e.g., antioxidants, citric acid, sodium citrate, benzalkonium chloride, chlorobutanol, cysteine, methionine, parabens, thimerosal), emulsifiers, buffer substances, flavoring agents, or coloring agents.

[0061] Salts are included in the present invention and these can also be pharmaceutically acceptable salts. Such salts or pharmaceutically acceptable salts include, but are not limited to, those made from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Salts or pharmaceutically acceptable salts can also be made as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts. Salts can be added to adjust the ionic strength or tonicity. Alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts. Salts can be added to adjust the ionic strength or tonicity.

[0062] Buffer substances suitable for use in the compositions according to the present invention or in pharmaceutical compositions include salts of acetic acid, salts of citric acid, salts of boric acid, and salts of phosphoric acid.

[0063] The composition or pharmaceutical composition can also be formulated as a stable lyophilized product to be reconstituted with a suitable diluent, which may optionally contain one or more excipients as defined above.

[0064] The active agents and pharmaceutical compositions described herein can be administered in a therapeutically effective amount. A "therapeutically effective amount" refers, in one embodiment, to an amount that achieves a desired therapeutic response or desired therapeutic effect, alone or in conjunction with further dosages, without causing unacceptable side effects. In the case of treating a particular disease or a particular condition, the desired response relates to inhibiting the progression of the disease. This includes slowing the progression of the disease and, in particular, interrupting or reversing the progression of the disease. The desired response in treating a disease or condition can also be delaying or preventing the onset of the disease or the condition. The effective amount of the agents and compositions described herein will be dependent on the condition to be treated, the severity of the disease, the individual parameters of the subject (including age, physiological state, size and weight), the duration of the treatment, the type of concomitant therapy (if any), the particular route of administration, and like factors. Accordingly, the dosage administered of the agents described herein may be dependent on several such parameters. If the response in the subject is not sufficient with the initial dosage, higher dosages (or, effectively higher dosages administered by a different route of administration that is more local) can also be used.

[0065] As used herein, the term "kit of parts (i.e., kit)" refers to a manufactured article that includes one or more containers and optionally a data medium. The one or more containers can hold one or more of the above-described agents (reagents). The kit can include, for example, additional containers containing diluents, buffers, and additional reagents. The data medium can be a non-electronic data medium, such as an information leaflet, information sheet, graphic data medium such as a barcode or access code, or an electronic data medium such as a compact disc (CD), digital versatile disc (DVD), microchip, or another semiconductor-based electronic data medium. The access code enables access to a database, such as an Internet database, centralized database, or distributed database. The data medium may include instructions for use of the active agent of the present invention. For example, the data medium may include instructions for use of the active agent (a) in a manufacturing method, particularly in a manufacturing method for producing a peptide, particularly in a manufacturing method for producing human insulin, insulin derivatives, and / or insulin analogs; (b) as a food ingredient; (c) as a feed ingredient; or (d) in a method for producing a food ingredient or feed ingredient.

[0066] As used herein, the term "preproinsulin" or the abbreviation "PPI" refers to a single-chain insulin precursor starting from the N-terminal portion, followed by a presequence and then a B-chain, C-peptide, and A-chain (see Figure 1).

[0067] Embodiments of the Invention In the following sections, certain elements of the present invention are described. It should be understood that these elements are listed with specific embodiments, but can be combined in any manner and in any number to create additional embodiments. The various examples described The embodiments should not be construed as limiting the present invention only to the explicitly described embodiments. This description is to be understood as supporting and encompassing embodiments that combine the explicitly described embodiments with any number of the disclosed elements. Further, any replacement and combination of all the described elements in this application should be regarded as disclosed by the description of this application unless otherwise specified in the context.

[0068] In a first aspect, the present invention is a variant of porcine trypsin, comprising or consisting of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, wherein the amino acid sequence differs from SEQ ID NO: 1 in that there is at least one or more amino acid substitutions at one or more positions corresponding to F24, S44, D56, G78, Y131, S172 and W193 of native porcine trypsin according to SEQ ID NO: 1, provided that the amino acid sequence is not native porcine trypsin according to SEQ ID NO: 1; and the amino acid sequence is not porcine mutant trypsin S172A according to SEQ ID NO: 2, said variant of porcine trypsin, is targeted.

[0069] In one embodiment of the first aspect, the amino acid at the position corresponding to F24 is not substituted with Asp, Glu, or Gly; the amino acid at the position corresponding to S44 is not substituted with Tyr; the amino acid at the position corresponding to Y131 is not substituted with Lys or Pro; and / or the amino acid at the position corresponding to W193 is not substituted with Asn or Cys.

[0070] In one embodiment of the first aspect, The amino acid at the position corresponding to F24 is substituted with an amino acid selected from the group consisting of Ala, Asn, Arg, Gln, Ile, Leu, Lys, Met, Ser, Thr, and Val; The amino acid at the position corresponding to S44 is substituted with an amino acid selected from the group consisting of Leu and Pro; The amino acid at the position corresponding to D56 is substituted with an amino acid selected from the group consisting of Ala, Asn, His, and Trp; The amino acid at the position corresponding to G78 is substituted with an amino acid selected from the group consisting of Ala, Glu, Pro, Ser, and Tyr; The amino acid at the position corresponding to Y131 is substituted with an amino acid selected from the group consisting of Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Ser, Thr, Trp, and Val; The amino acid at the position corresponding to S172 is substituted with an amino acid selected from the group consisting of Ala, Cys, and Thr; and / or The amino acid at the position corresponding to W193 is substituted with an amino acid selected from the group consisting of Phe, Ser, Thr, and Tyr.

[0071] In a further embodiment of the first aspect, The amino acid at the position corresponding to F24 is substituted with Arg; The amino acid at the position corresponding to S44 is substituted with Pro; The amino acid at the position corresponding to D56 is substituted with His; The amino acid at the position corresponding to G78 is substituted with Pro; The amino acid at the position corresponding to Y131 is substituted with Met; The amino acid at the position corresponding to S172 is substituted with Ala; and / or The amino acid at the position corresponding to W193 is substituted with Ser.

[0072] In one embodiment of the first aspect, the amino acid sequence further differs from SEQ ID NO: 1 in that there is at least one or more amino acid substitutions at one or more positions corresponding to R99, R107, K125, and K170 of native porcine trypsin according to SEQ ID NO: 1.

[0073] In one embodiment of the first aspect, the amino acid at the position corresponding to R107 is not substituted with Pro. And / or the amino acid at the position corresponding to K170 is not substituted with Ile or Phe.

[0074] In a further embodiment of the first aspect, the amino acid at the position corresponding to R99 is substituted with an amino acid selected from the group consisting of Ala, Asn, Asp, Glu, Gly, His, Leu, Phe, Thr, Trp, and Tyr; the amino acid at the position corresponding to R107 is substituted with an amino acid selected from the group consisting of Asp, Gly, Pro, Ser, and Thr; the amino acid at the position corresponding to K125 is substituted with an amino acid selected from the group consisting of Ala, Cys, Gln, Glu, Gly, His, Leu, Ser, and Tyr; and / or the amino acid at the position corresponding to K170 is substituted with an amino acid selected from the group consisting of Ala, Asn, Gly, and Tyr.

[0075] In a further embodiment of the first aspect, the amino acid at the position corresponding to R99 is substituted with an amino acid selected from the group consisting of Ala, His, and Asn; the amino acid at the position corresponding to R107 is substituted with Thr; the amino acid at the position corresponding to K125 is substituted with an amino acid selected from the group consisting of Ala, Cys, and Ser; and / or the amino acid at the position corresponding to K170 is substituted with Ala.

[0076] In one embodiment of the first aspect, the amino acid sequence has at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% sequence identity with porcine wild-type trypsin according to SEQ ID NO: 1.

[0077] In one embodiment of the first aspect, the amino acid sequence differs from porcine wild-type trypsin according to SEQ ID NO: 1 in that there are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acid substitution mutations.

[0078] In one embodiment of the first aspect, the amino acid sequence is SEQ ID NO: 3, where Xaa24 is an amino acid selected from the group consisting of Ala, Asn, Arg, Gln, Ile, Leu, Lys, Met, Phe, Ser, Thr, and Val; Xaa44 is an amino acid selected from the group consisting of Leu, Pro, and Ser; Xaa56 is an amino acid selected from the group consisting of Ala, Asn, Asp, His, and Trp selected; Xaa78 is an amino acid selected from the group consisting of Ala, Glu, Gly, Pro, Ser, and Tyr; Xaa99 is an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Glu, Gly, His, Leu, Phe, Thr, Trp, and Tyr; Xaa107 is an amino acid selected from the group consisting of Arg, Asp, Gly, Pro, Ser, and Thr; Xaa125 is an amino acid selected from the group consisting of Ala, Cys, Gln, Glu, Gly, His, Leu, Lys, Ser, and Tyr; Xaa131 is an amino acid selected from the group consisting of Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr, and Val; Xaa170 is an amino acid selected from the group consisting of Ala, Asn, Gly, Lys, and Tyr; Xaa172 is an amino acid selected from the group consisting of Ala, Cys, Ser, and Thr; and / or Xaa193 is an amino acid selected from the group consisting of Phe, Ser, Thr, Trp, and Tyr; provided that SEQ ID NO: 3 is not the porcine wild-type trypsin according to SEQ ID NO: 1; and SEQ ID NO: 3 is not the porcine mutant trypsin S172A according to SEQ ID NO: 2.

[0079] In a further embodiment of the first aspect, the amino acid sequence is SEQ ID NO: 3, where Xaa24 is an amino acid selected from the group consisting of Phe and Arg; Xaa44 is an amino acid selected from the group consisting of Ser and Pro; Xaa56 is an amino acid selected from the group consisting of Asp and His; Xaa78 is an amino acid selected from the group consisting of Gly and Pro; Xaa99 is an amino acid selected from the group consisting of Ala, Arg, His, and Asn; Xaa107 is an amino acid selected from the group consisting of Arg and Thr; Xaa125 is an amino acid selected from the group consisting of Ala, Cys, Lys and Ser; Xaa131 is an amino acid selected from the group consisting of Met and Tyr; Xaa170 is an amino acid selected from the group consisting of Ala and Lys; Xaa172 is an amino acid selected from the group consisting of Ala and Ser; and / or Xaa193 is an amino acid selected from the group consisting of Ser and Trp; provided that SEQ ID NO: 3 is not porcine wild-type trypsin according to SEQ ID NO: 1; and SEQ ID NO: 3 is not porcine mutant trypsin S172A according to SEQ ID NO: 2.

[0080] In one embodiment of the first aspect, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 4 to 119. In one embodiment, the amino acid sequence is selected from the group consisting of SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98.

[0081] In one embodiment of the first aspect, the variant can cleave a peptide having the general formula A-Lys-Thr-Arg-Arg-B to yield a cleavage product of the general formula A-Lys-Thr-Arg-Arg with a yield of at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%); A is an amino acid sequence consisting of one or more amino acids; and B is an amino acid sequence consisting of one or more amino acids.

[0082] In a further embodiment of the first aspect, A is an amino acid sequence consisting of 1 to 105 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 105 amino acids), and B is an amino acid sequence consisting of 1 to 105 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 105 amino acids).

[0083] In one embodiment of the first aspect, the variant exhibits improved selectivity for the cleavage of preproinsulin, preproinsulin glyralgin, preproinsulin lispro, preproinsulin asparto, or preproproinsulin glyrlys at a position on the C-terminal side of the B32-Arg position as compared to porcine mutant trypsin S172A according to SEQ ID NO: 2.

[0084] In some embodiments of the first aspect of the present invention, the variant of porcine trypsin is lyophilized.

[0085] In the second aspect, the present invention is directed to a nucleic acid molecule comprising - the variant of porcine trypsin as defined in the first aspect of the present invention; or - the trypsinogen precursor molecule of the variant of porcine trypsin as defined in the first aspect of the present invention and encoding the nucleic acid molecule.

[0086] In one embodiment of the second aspect, the nucleic acid molecule is contained in a vector or integrated into the genome.

[0087] In a third aspect, the present invention is directed to a host cell containing the nucleic acid molecule defined in the second aspect. In one embodiment, the host cell is a recombinant host cell.

[0088] In a fourth aspect, the present invention is a method for producing a variant of porcine trypsin comprising: culturing the host cell according to the third aspect; and isolating the trypsinogen precursor molecule of the variant of porcine trypsin from the culture medium or from the host cell is directed to.

[0089] In one embodiment of the fourth aspect, the method further comprises the step of: activating the trypsinogen precursor molecule to thereby obtain the variant of porcine trypsin is included.

[0090] The activation of the trypsinogen precursor molecule can be carried out chemically or enzymatically. For example, the pH can be adjusted so that autoactivation occurs. In another example, a protease (such as enterokinase) can be added to enzymatically activate trypsinogen.

[0091] In a fifth aspect, the present invention is for use as a medicament; for use as a food ingredient; for use as a feed ingredient, or for use in a method for producing a food ingredient or a feed ingredient, the variant of porcine trypsin of the present invention, or the nucleic acid molecule of the present invention, or the host cell of the present invention is directed to.

[0092] One embodiment of the fifth aspect is directed to the variant of porcine trypsin of the present invention for use as a food ingredient; for use as a feed ingredient, or for use in a method for producing a food ingredient or a feed ingredient.

[0093] In one embodiment of the fifth aspect, the medicament is for use in the treatment of swelling (especially swelling caused by trauma), inflammation (especially inflammation caused by trauma), thrombophlebitis, activated arthrosis, deficiency of digestive enzymes (e.g., caused by pancreatic insufficiency), and / or digestive disorders.

[0094] In other words, the present invention is directed to the use of a variant of porcine trypsin of the present invention, a nucleic acid molecule of the present invention, or a host cell of the present invention for use in the treatment of swelling (especially swelling caused by trauma), inflammation (especially inflammation caused by trauma), thrombophlebitis, activated arthrosis, deficiency of digestive enzymes (e.g., caused by pancreatic insufficiency), and / or digestive disorders.

[0095] In relation to use as a food ingredient, as a feed ingredient, or in the context of a manufacturing method, the fifth aspect can alternatively be expressed as follows: Use of a variant of porcine trypsin of the present invention as a food ingredient. Use of a variant of porcine trypsin of the present invention as a feed ingredient. Use of a variant of porcine trypsin of the present invention in a method for producing a food ingredient or a feed ingredient.

[0096] In the sixth aspect, the present invention is directed to the use of a variant of porcine trypsin of the present invention in a method for producing human insulin, an insulin analog, or a derivative of insulin.

[0097] The sixth aspect encompasses the use of a variant of porcine trypsin of the present invention in both in vitro and in vivo methods for producing human insulin, an insulin analog, or a derivative of insulin.

[0098] In relation to use in an in vivo method, the sixth aspect can alternatively be expressed as follows: The variant of porcine trypsin described in the first aspect for use in the in vivo production of human insulin, insulin analogs or derivatives of insulin. In one embodiment of the sixth aspect, the insulin analog is selected from the group consisting of insulin aspart, insulin lispro, insulin glulisine, and insulin glargine.

[0099] In the seventh aspect, the present invention has the general formula A-Lys-Thr-Arg-Arg-B Use of the variant of porcine trypsin of the present invention for cleaving a protein or peptide, wherein A is an amino acid sequence consisting of one or more amino acids; and B is an amino acid sequence consisting of one or more amino acids, for the said use.

[0100] In a further embodiment of the seventh aspect, A is an amino acid sequence consisting of amino acids between 1 and 105 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 105 amino acids)), and B is an amino acid sequence consisting of amino acids between 1 and 105 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 105 amino acids).

[0101] In one embodiment of the seventh aspect, cleavage results in the cleavage product A-Lys-Thr-Arg-Arg. In a further embodiment of the seventh aspect, cleavage results in the cleavage product A-Lys-Thr-Arg-Arg in a yield of at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%).

[0102] In the eighth aspect, the present invention is directed to a composition comprising a variant of porcine trypsin of the present invention, a nucleic acid molecule of the present invention, or a host cell of the present invention.

[0103] In one embodiment of the eighth aspect, the composition further comprises an excipient and / or at least one other active agent.

[0104] In some embodiments of the eighth aspect, the composition is a pharmaceutical composition.

[0105] In some embodiments of the eighth aspect, the composition or the pharmaceutical composition is lyophilized.

[0106] The present invention also provides a combination of a variant of porcine trypsin of the present invention (or a nucleic acid molecule of the present invention or a host cell of the present invention) and at least one other active pharmaceutical ingredient. In one embodiment, the combination of the variant of porcine trypsin of the present invention and at least one other active pharmaceutical ingredient can be applied either by separate administration of the active pharmaceutical ingredients to a patient or in the form of a combination product in which the plurality of active pharmaceutical ingredients are present in one pharmaceutical composition. When administered separately, the administrations may be carried out simultaneously or sequentially in any order. For the purpose of achieving the desired combined therapeutic effect, the amounts of the active agent of the present invention and the other active pharmaceutical ingredients and the relative timing of administration will be selected. The administration of the combination may be concomitant with (1) a single pharmaceutical composition containing all the active pharmaceutical ingredients; or (2) individual pharmaceutical compositions each containing at least one of the active pharmaceutical ingredients. Alternatively, the combination can be administered separately in sequence, in which case one therapeutic agent is administered first and the other is administered second, and vice versa. Such sequential administrations may be close in time or separated in time.

Example

[0107] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the compounds, compositions, and methods of the present invention, and are not intended to limit the scope of the present invention in any way as defined by the appended claims. Although efforts have been made to be accurate with respect to the numerical values used (e.g., amounts, temperatures, etc.), some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

Example

[0108] Mutagenesis and cloning of a synthetic nucleotide sequence encoding porcine trypsin Improvements to enzymes can be made by enzyme engineering. This technique involves the development of variants of a starting enzyme with improved properties. To provide enzyme variants, mutations were first introduced at the gene level into a synthetic nucleotide sequence encoding porcine pancreatic trypsin by conventional molecular biology methods. The gene for the enzyme variant was cloned into the expression vector pLE1A17 (a derivative of pRSF-1b, Novagen). The resulting plasmid was used for the transformation of Escherichia coli BL21(DE3) cells.

Example

[0109] Expression of variant porcine trypsinogen Recombinant expression of porcine trypsinogen in E. coli was carried out by autoinduction at 30 °C using medium ZYM-5052 supplemented with kanamycin (50 mg / L) according to F.W. Studier (F.W. Studier (2005) Prot. Exp. Pur. 41: 207-234). When the glucose present during the culture is metabolized, the lactose present induces expression. Glycerol was used as the C source. Expression was carried out both in microplates and at the shake flask scale.

[0110] For the shake flask experiment, a preculture with 20 mL of medium ZYM-5052 supplemented with kanamycin (50 mg / L) containing 50 μL of glycerol (36%) was incubated at 30 °C and 200 rpm for 24 hours. An OD600 of 12.5 - 14 was obtained. For the main culture, 500 μL of the preculture was used to inoculate 100 mL of medium ZYM-5052 supplemented with kanamycin (50 mg / L). After incubation at 30 °C and 200 rpm for 24 hours, an OD600 of 6.5 - 7.5 was obtained. After centrifugation, the cell pellet was stored at -20 °C.

Example

[0111] Cell disruption and trypsinogen activation Periplasmatic trypsinogen was released by osmotic shock. 3.6 g of cells (obtained from Example 2) were resuspended in 10 mL of lysis buffer (10 mM Tris-HCl, pH = 8, 25 mM MgCl2, 200 mM NaCl) until a uniform suspension was obtained. The suspension was stirred at 5 °C and 180 rpm for 24 hours. After centrifugation, the supernatant was subjected to activation. For the activation of trypsinogen, enterokinase derived from porcine intestine was used (Sigma, product no. E0885; 40 units; definition of unit: 1 unit of enterokinase produces 1.0 nanomole of trypsin from trypsinogen per minute at 25 °C and pH 5.6). Enterokinase was added to the cell-free extract to a final concentration of 17 mU / mL. The reaction was incubated at 30 °C for 16 - 24 hours. The resulting trypsin solution was stored at -20 °C.

Example

[0112] Assay for determining the trypsin activity of purified recombinant porcine trypsin, a mutant Trypsin activity was determined at 25 °C using Chromozym TRY (Roche Diagnostics GmbH) contained in 100 mM Tris pH 8.0, 20 mM CaCl2. Chromozym TRY is a reliable substrate for absorbance spectrophotometric quantification of the activity of proteases (trypsin, endoproteinase, Arg-C, etc.) that hydrolyze peptides on the carboxylic acid side of arginine (carbobenzoxy-L-valyl-L-glycyl-L-arginine-4-nitroanilide acetate). Spectrophotometric measurements were performed at 405 nm.

Example

[0113] Screening results with preproinsulin. The primary screening of all trypsin mutants was performed using preproinsulin (PPI) as a model system. PPI was dissolved in a buffer solution (pH = 8.3) to a concentration of 0.75 g / L. The trypsin mutant solution was prepared according to Example 3 and added to the reaction mixture without further treatment. Samples were taken periodically, acidified with 1N HCl, and analyzed by HPLC (Example 7).

[0114] Two aspects were considered: (1) Whether the trypsin mutant exhibits any PPI cleavage activity (results shown in Table 1, column A); (2) Whether the trypsin mutant exhibits PPI cleavage activity: whether the trypsin cleavage selectivity is improved compared to SEQ ID NO: 2 (results shown in Table 1, column B). To evaluate the di-Arg selectivity of the trypsin mutant, the formation of B31Arg-B32Arg-human insulin was monitored by HPLC (see Example 7) and compared to SEQ ID NO: 2. The selectivity was [Number] calculated by. Here, [diArg] = concentration of B31Arg-B32Arg-human insulin [monoArg] = concentration of desB32Arg-human insulin [desThr] = concentration of desB30Thr-human insulin [UI] = concentration of unknown cleavage intermediate is.

[0115] [Table 1] [Table 2] [Table 3] [Example]

[0116] Cleavage of preproinsulin gralgin using recombinant SEQ ID NO:2 (trypsin variant S172A) and its optimized variants This experiment was carried out at 20 °C, pH 8.3 (buffer solution) and up to a 1000 mL scale. The preproinsulin gralgin solution was filled into a suitable thermostatic reaction vessel, and the reaction was initiated by adding a solution of the porcine trypsin variant. Samples were taken after certain time intervals; the enzyme reaction was immediately stopped by acidifying the sample solution using 1 N or 2 N HCl solution. The samples were analyzed by HPLC (see Example 7). Figure 2 shows the kinetics of the reaction of SEQ ID NO:2. The figure shows the time-dependent course of insulin gralgin formation, as well as the most prominent cleavage by-product B31Arg-insulin gralgin ( cleavage after position 31 of the insulin gralgin B chain, called "des-Arg") and the formation of desB30Thr-insulin gralgin (cleavage after position 29 of the insulin gralgin B chain, called "des-Thr"). The selected variants were subjected to the cleavage of preproinsulin gralgin. The results are shown in Table 2. The contents of insulin gralgin, desB32Arg-insulin gralgin, and desB29Thr-insulin gralgin are given in the form of peak areas from the analytical HPLC chromatogram at the end of the cleavage, representing the ratio of insulin gralgin to by-products. Furthermore, the molar cleavage yield is [Number] given according to n(insulin gralgin) = moles of insulin gralgin formed n(preproinsulin gralgin) = moles of preproinsulin gralgin deployed where.

[0117] Figure 4 shows the kinetics of the porcine trypsin variant no. 105 (SEQ ID NO:105) as an example of the optimized trypsin variant.

[0118]

Table 4

Table 5

[0119] As is clearly shown in Table 2, the optimized trypsin variants showed improved cleavage selectivity for preproinsulin glucagon, resulting in a significant improvement in yield, which, considering B31Arg-insulin glucagon (desArg) and desB30Thr-insulin glucagon (desThr), was accompanied by a reduced impurity profile. Comparing Table 1 and Table 2, it is shown that there was a very high similarity in the cleavage results obtained with preproinsulin human and preproinsulin glucagon.

Examples

[0120] HPLC method Method High Performance Liquid Chromatography Column Manufacturer: Macherey & Nagel Brand: Nucleosil 120-5 C18 Dimensions: 250×4 mm Mobile phase A: 45 mM sodium phosphate buffer (pH 2.5), 315 mM NaCl. 25% (v / v) acetonitrile Mobile phase B 45 mM sodium phosphate buffer (pH 2.5), 55 mM NaCl. 65% (v / v) acetonitrile Gradient Time / min Mobile phase A Mobile phase B 0 min 94% 6% Linear for 30 min 90% 10% Injection volume: 5 μL Detection: UV (215 nm)

Examples

[0121] Exopeptidase activity This experiment was carried out at 8 °C and pH value 8.3 (buffer solution). Fill an appropriate constant-temperature reaction vessel with an insulin glargine solution (0.5 g / L), and start the reaction by adding the solution of SEQ ID NO: 2 (100 U / g インスリン ). Samples were taken after certain time intervals, and the enzyme reaction was immediately stopped by acidifying the sample solution using 1N HCl solution. The samples were analyzed by HPLC (see Example 7). The reaction kinetics is shown in Figure 3.

[0122] As clearly shown in Figure 3, the concentration of insulin glargine decreases over time. In addition to the formation of a small amount of B30-desThr-insulin glargine, the main product formed in this reaction is B32-desArg-insulin glargine, indicating that trypsin SEQ ID NO: 2 can cleave the single arginine at position B32 by exopeptidase activity.

[0123] [Table 6] [Table 7] [Table 8] [Table 9]

[0124] Full-length sequences of wild-type porcine trypsin and selected trypsin mutants Wild-type porcine trypsin, SEQ ID NO: 1: IVGGYTCAAN SIPYQVSLNS GSHFCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DSCQGDSGGP VVCNGQLQGI VSWGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Variant S172A, SEQ ID NO: 2 IVGGYTCAAN SIPYQVSLNS GSHFCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSWGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Gene sequence of porcine trypsin with variable positions at amino acid positions 24, 44, 56, 78, 99, 107, 125, 131, 170, 172, and 193; SEQ ID NO: 3 IVGGYTCAAN SIPYQVSLNS GSHXCGGSLI NSQWVVSAAH CYKXRIQVRL GEHNIXVLEG NEQFINAAKI ITHPNFNXNT LDNDIMLIKL SSPATLNSXV ATVSLPXSCA AAGTECLISG WGNTXSSGSS XPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGX DXCQGDSGGP VVCNGQLQGI VSXGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN SEQ ID NO: 4 IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSAV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 5: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSLV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 6: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSFV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 7: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSWV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 8: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSGV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 9: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSYV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 10: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSTV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 11: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSNV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 12: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSDV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 13: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSEV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN SEQ ID NO: 14 IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSHV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN SEQ ID NO: 15 IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTASSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN SEQ ID NO: 16 IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTGSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 17: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTYSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 18: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTSSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 19: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTCSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 20: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTESSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 21: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTHSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 22: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTLSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 23: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTQSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 24: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGY DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 25: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 26: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGG DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 27: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGN DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 28: IVGGYTCAAN SIPYQVSLNS GSHVCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 29: IVGGYTCAAN SIPYQVSLNS GSHLCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 30: IVGGYTCAAN SIPYQVSLNS GSHSCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 31: IVGGYTCAAN SIPYQVSLNS GSHQCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 32: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 33: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPTSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 34: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS APSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN SEQ ID NO: 35: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS LPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN SEQ ID NO: 36: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN SEQ ID NO: 37: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS TPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 38: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS CPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 39: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS NPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 40: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS EPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 41: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS HPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 42: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 43: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DTCQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 44: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DSCQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 45: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DCCQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 46: IVGGYTCAAN SIPYQVSLNS GSHACGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 47: IVGGYTCAAN SIPYQVSLNS GSHICGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 48: IVGGYTCAAN SIPYQVSLNS GSHMCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 49: IVGGYTCAAN SIPYQVSLNS GSHTCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 50: IVGGYTCAAN SIPYQVSLNS GSHNCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 51: IVGGYTCAAN SIPYQVSLNS GSHKCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 52: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKLRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 53: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 54: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIAVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 55: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIWVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 56: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNINVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 57: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 58: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPPSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 59: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPGSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 60: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPSSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 61: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPDSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 62: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS VPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 63: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS IPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 64: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS WPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 65: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS GPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 66: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS SPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 67: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS QPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 68: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS DPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 69: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSFGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 70: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSYGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 71: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSTGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 72: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNSNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 73: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNANT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 74: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 75: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNYNT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 76: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNENT LDNDIMLIKL SSPATLNSRV ATVSLPRSCA AAGTECLISG WGNTKSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 77: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSHV ATVSLPTSCA AAGTECLISG WGNTCSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 78: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPTSCA AAGTECLISG WGNTCSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 79: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPRSCA AAGTECLISG WGNTASSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 80: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPTSCA AAGTECLISG WGNTSSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 81: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPTSCA AAGTECLISG WGNTASSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 82: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPRSCA AAGTECLISG WGNTCSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 83: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSNV ATVSLPTSCA AAGTECLISG WGNTSSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 84: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSNV ATVSLPTSCA AAGTECLISG WGNTSSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DSCQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 85: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPTSCA AAGTECLISG WGNTCSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 86: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSAV ATVSLPTSCA AAGTECLISG WGNTSSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 87: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPTSCA AAGTECLISG WGNTSSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 88: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPTSCA AAGTECLISG WGNTSSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DSCQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 89: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPTSCA AAGTECLISG WGNTASSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 90: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPTSCA AAGTECLISG WGNTASSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DSCQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 91: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSAV ATVSLPTSCA AAGTECLISG WGNTSSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 92: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSAV ATVSLPTSCA AAGTECLISG WGNTCSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 93: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSNV ATVSLPTSCA AAGTECLISG WGNTCSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 94: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSNV ATVSLPRSCA AAGTECLISG WGNTCSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN SEQ ID NO: 95: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPRSCA AAGTECLISG WGNTSSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN SEQ ID NO: 96: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPRSCA AAGTECLISG WGNTSSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN SEQ ID NO: 97: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSAV ATVSLPTSCA AAGTECLISG WGNTASSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 98: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSAV ATVSLPTSCA AAGTECLISG WGNTASSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DSCQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 99: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSNV ATVSLPTSCA AAGTECLISG WGNTSSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 100: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPTSCA AAGTECLISG WGNTCSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN SEQ ID NO: 101: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSNV ATVSLPTSCA AAGTECLISG WGNTSSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN SEQ ID NO: 102: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPTSCA AAGTECLISG WGNTSSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN SEQ ID NO: 103: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPTSCA AAGTECLISG WGNTSSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DSCQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 104: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSNV ATVSLPTSCA AAGTECLISG WGNTCSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 105: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSNV ATVSLPTSCA AAGTECLISG WGNTASSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 106: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSAV ATVSLPTSCA AAGTECLISG WGNTCSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 107: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPTSCA AAGTECLISG WGNTCSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 108: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIDVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSAV ATVSLPTSCA AAGTECLISG WGNTCSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 109: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSNV ATVSLPTSCA AAGTECLISG WGNTCSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 110: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKSRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPRSCA AAGTECLISG WGNTCSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 111: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPTSCA AAGTECLISG WGNTSSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 112: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNPNT LDNDIMLIKL SSPATLNSHV ATVSLPTSCA AAGTECLISG WGNTSSSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DSCQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 113: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSAV ATVSLPTSCA AAGTECLISG WGNTASSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 114: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSAV ATVSLPTSCA AAGTECLISG WGNTASSGSS YPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DSCQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 115: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSNV ATVSLPTSCA AAGTECLISG WGNTSSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 116: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSNV ATVSLPTSCA AAGTECLISG WGNTSSSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 117: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSNV ATVSLPTSCA AAGTECLISG WGNTASSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 118: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSHV ATVSLPTSCA AAGTECLISG WGNTASSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGA DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN Sequence number 119: IVGGYTCAAN SIPYQVSLNS GSHRCGGSLI NSQWVVSAAH CYKPRIQVRL GEHNIHVLEG NEQFINAAKI ITHPNFNGNT LDNDIMLIKL SSPATLNSAV ATVSLPTSCA AAGTECLISG WGNTASSGSS MPSLLQCLKA PVLSDSSCKS SYPGQITGNM ICVGFLEGGK DACQGDSGGP VVCNGQLQGI VSSGYGCAQK NKPGVYTKVC NYVNWIQQTI AAN

Claims

1. A variant of porcine trypsin, comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, said variant of porcine trypsin.

2. (i) The variant is capable of cleaving a peptide having the general formula A-Lys-Thr-Arg-Arg-B to yield a cleavage product of the general formula A-Lys-Thr-Arg-Arg in a yield of at least 80%, wherein A is an amino acid sequence consisting of one or more amino acids; and B is an amino acid sequence consisting of one or more amino acids; and / or (ii) The variant exhibits improved selectivity for cleavage of preproinsulin, preproinsulin gralargin, preproinsulin lispro, preproinsulin aspart, or preproinsulin glulys at a position on the C-terminal side of the B32-Arg position as compared to porcine variant trypsin S172A according to SEQ ID NO: 2, The variant of porcine trypsin according to claim 1.

3. A nucleic acid molecule, - encoding the variant of porcine trypsin according to claim 1 or 2, or - a trypsinogen precursor molecule of the variant of porcine trypsin according to claim 1 or 2, said nucleic acid molecule optionally contained in a vector or optionally integrated into the genome.

4. A host cell containing the nucleic acid molecule according to claim 3.

5. A method for producing the variant of porcine trypsin according to claim 1 or 2, comprising: culturing the host cell according to claim 4; and isolating the trypsinogen precursor molecule of the variant of porcine trypsin from the culture medium or from the host cell, and optionally activating the trypsinogen precursor molecule to thereby obtain the variant of porcine trypsin.

6. The variant of porcine trypsin according to claim 1 or 2, the nucleic acid molecule according to claim 3, or the host cell according to claim 4 for use in a method for producing a food ingredient or a feed ingredient.

7. Use of the variant of porcine trypsin according to claim 1 or 2 in a method for the production of human insulin, an insulin analog, or a derivative of insulin.

8. ​ ​ ​ ​ The use according to claim 7, wherein the insulin analog is selected from the group consisting of insulin aspart, insulin lispro, insulin glulisine, and insulin glargine.

9. Use of a variant of porcine trypsin according to claim 1 or 2 for cleaving a protein or peptide having the general formula A-Lys-Thr-Arg-Arg-B, wherein A is an amino acid sequence consisting of one or more amino acids; and B is an amino acid sequence consisting of one or more amino acids.

10. Use according to claim 9, wherein cleavage results in the cleavage product A-Lys-Thr-Arg-Arg.

11. Use according to claim 10, wherein cleavage results in the cleavage product A-Lys-Thr-Arg-Arg in a yield of at least 80%.

Images