CN111072783A - Method for preparing GLP-1 or analog polypeptide thereof by adopting escherichia coli expression tandem sequence - Google Patents

Method for preparing GLP-1 or analog polypeptide thereof by adopting escherichia coli expression tandem sequence Download PDF

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CN111072783A
CN111072783A CN201911373533.3A CN201911373533A CN111072783A CN 111072783 A CN111072783 A CN 111072783A CN 201911373533 A CN201911373533 A CN 201911373533A CN 111072783 A CN111072783 A CN 111072783A
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CN111072783B (en
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辛中帅
赵珊珊
文良柱
平康康
徐晓峰
李夷平
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Jiangsu Wanbang Pharmaceutical Technology Co ltd
Wanxin Pharmaceutical Technology Suzhou Co Ltd
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Wanxin Pharmaceutical Technology Suzhou Co Ltd
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Abstract

The invention discloses a method for preparing GLP-1 or analog polypeptide thereof by adopting escherichia coli expression tandem sequence, and the general formula of recombinant tandem protein of GLP-1 or analog thereof designed by the invention is as follows: x- (Y-Z) n. Wherein: x is leader peptide, Y is trypsin and CPB enzyme double restriction site or Kex2 enzyme and CPB enzyme double restriction site and auxiliary sequence, and Z is GLP-1 or its analogue. n is the number of series repeats. The inclusion body formed by recombinant tandem protein for fermenting, inducing and expressing GLP-1 or the analogue thereof through an escherichia coli prokaryotic expression system can be completely dissolved without a denaturant, and GLP-1 or the analogue thereof is obtained by double enzyme digestion of trypsin and CPB enzyme or double enzyme digestion of Kex2 enzyme and CPB enzyme in a dissolving solution. The GLP-1 or the analog thereof obtained by the method has high expression level, and the GLP-1 target protein after enzyme digestion has high yield and purity after enzyme digestion.

Description

Method for preparing GLP-1 or analog polypeptide thereof by adopting escherichia coli expression tandem sequence
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for preparing GLP-1 or analog polypeptide thereof by adopting a series sequence efficiently expressed by escherichia coli.
Background
Type 2 diabetes is a highly heterogeneous metabolic disorder syndrome, the basic pathological impairment of which is a marked decline in islet function combined with insulin resistance. According to statistics, about 2.46 hundred million diabetics are in the world in 2007, and about 3.8 hundred million diabetics will be in 2025, wherein the number of the 2-type diabetics accounts for 90-95% of the total number. At present, the clinical treatment of diabetes mainly focuses on various insulin and four major oral hypoglycemic drugs. In order to provide more choices for diabetic patients during treatment, some novel diabetes treatment drugs, such as glucagon-like peptide-1 analogues, are produced at the beginning of the 21 st century, and the drugs are prepared by structural change on the basis of endogenous glucagon-like peptide-1 (GLP-1). Endogenous GLP-1 promotes insulin secretion as required by the glucose level in vivo, but is rapidly degraded by dipeptidyl peptidase IV in vivo with a half-life of about 2 min. The GLP-1 preparation with the modified structure not only retains the pharmacological action of GLP-1, but also greatly improves the action time of the medicament, and GLP-1 analogues approved by FDA for treating type 2 diabetes at present comprise exenatide injection, liraglutide injection, somaglutide tablet and the like.
GLP-1 or an analog thereof usually has a molecular weight of not more than 4000Da and an amino acid number of not more than 40, and can be produced by an Escherichia coli expression system and a yeast expression system. The yeast expression can be secreted outside cells, which is beneficial to the collection and purification of expression products, but the yeast culture cost is relatively high, the fermentation culture period time is long, a powerful promoter which is strictly regulated and controlled is lacked, the secretion efficiency is low, the relative yield is not high, and the comprehensive cost is high. The prokaryotic expression system of the escherichia coli has the characteristics of high yield, low cost, clear genetic background, clear characteristics, simple operation and the like, and can make up for the defects of a yeast expression system. However, the prokaryotic expression system of escherichia coli is not suitable for expressing polypeptide or protein with smaller molecular weight, because the content of small peptide and protein products expressed in escherichia coli is easy to degrade, and in order to enhance the stability of the small protein polypeptide, a section of fusion protein is often required to be connected during design so as to reduce the degradation of the small protein polypeptide. The connected fusion protein needs to be removed by protease enzyme digestion before later purification, so that the target protein accounts for not high amount of total protein after the fusion protein is subjected to enzyme digestion, most of the expression products are removed after enzyme digestion, and as a result, the theoretical yield of the target protein is only 20-30% even if the expression amount of the fusion protein is high. GLP-1 or its analogue has small molecular weight, and in order to increase its expression level in Escherichia coli, a conventional expression strategy is to add a fusion protein to the front end or the rear end of GLP-1 or its analogue to increase its protein size. The fusion protein-GLP-1 or the analog thereof or the GLP-1 or the analog thereof-fusion protein releases GLP-1 or the analog thereof after being enzyme-digested by protease, enterokinase or the like. The yield of GLP-1 or analogues thereof obtained by the expression design is not high, the theoretical yield is only about 25%, the product purity is low and is generally only 40-50%, the subsequent separation and purification are difficult, and the yield is low. Therefore, the exploration of a new GLP-1 or analogue high-expression technology has very significant meaning for developing new drugs and reducing production cost.
At present, no patent or literature about polypeptide or protein tandem expression technology exists, and one reason may be that the enzyme cutting sites among the tandem expression target proteins are difficult to be completely identified, so that the enzyme cutting is incomplete, or residual amino acids exist after the enzyme cutting, so that the yield of the target proteins is low.
Therefore, the need in the art is to provide a design for GLP-1 or its analogue recombinant tandem protein expression and a construction method of prokaryotic expression strain to improve the ratio of GLP-1 or its analogue in the whole expression peptide chain and the yield after enzyme digestion.
Disclosure of Invention
One of the objects of the present invention is to provide recombinant tandem protein sequences of GLP-1 or analogs thereof.
Another object of the present invention is to provide a method for constructing a prokaryotic expression strain of recombinant tandem proteins of GLP-1 or analogues thereof.
The term "GLP-1 or analog thereof" as used herein means all or part of a mutated sequence of the native GLP-1 amino acid sequence, including but not limited to any one of the following (1) to (8):
(1)HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG、
(2)HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG、
(3)EGTFTSDVSSYLEGQAAKEFIAWLVRGRG、
(4)HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS、
(5)HAEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS、
(6)HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK、
(7)HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR、
(8)HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG。
in a first aspect of the invention, there is provided a recombinant tandem protein sequence of GLP-1 or an analog thereof, having the general formula: x- (Y-Z) n, wherein:
x is a leader peptide formed from 5-10 amino acids, the amino acid composition including but not limited to: alanine A, leucine L, N asparagine, R arginine, S serine, F phenylalanine, H histidine, M methionine, V valine. The preferred sequence composition is MRLN, MRLNSA or MRLNSAFV, and the sequence numbers are Seq ID No.1-3 respectively;
y is a core sequence of a tandem expression sequence design technology, and the general formula is Y-m-Y; wherein y is an enzyme cleavage site, and the amino acid composition includes but is not limited to: r arginine, K lysine, can be R or K or RR or KK or RK or KR, m is a proprietary linker sequence formed by 4-12 amino acids including but not limited to the combination of S serine, G glycine, E glutamic acid, D aspartic acid, T threonine, R arginine, A alanine; preferred sequence compositions are GSGSEEGSGS or GSGSDDGSGS or GSGSEDGSGS or GSGSDEGSGS or EEAE or DDAD or TEEAEKL or TDDADKL or TEDAEKL or TDEADKL or EGTEEAEKLG or EGTDDADKLG or EGTEEADKLG or EGTDDAEKLG or EGTEDADKLG or EGTDEAEKLG, with the sequence numbers Seq ID No.4-19, respectively;
z is GLP-1 or an analogue thereof, and the amino acid sequence is selected from any one of Seq ID No.20 to Seq ID No. 27:
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG(Seq ID No.20)、
HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG(Seq ID No.21)、
EGTFTSDVSSYLEGQAAKEFIAWLVRGRG(Seq ID No.22)、
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(Seq ID No.23)、
HAEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(Seq ID No.24)、
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK(Seq ID No.25)、
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR(Seq ID No.26)、
HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG(Seq ID No.27)；
n is a number of repeats, including but not limited to an integer from 2 to 7, preferably 3, 4 or 5.
In some embodiments, the invention provides specific GLP-1 or analog recombinant tandem protein sequences, and the amino acid sequence is shown as SEQ ID NO.28-SEQ ID NO. 111.
The invention also provides a gene for coding the GLP-1 or analogue recombinant tandem protein sequence thereof.
The invention also provides a recombinant plasmid containing a gene encoding the GLP-1 or analogue recombinant tandem protein sequence thereof.
In another preferred embodiment, the recombinant plasmid of the present invention comprises the gene of the present invention encoding the recombinant tandem protein sequence of GLP-1 or its analog, and a vector constituting the recombinant plasmid, preferably a pET series vector.
The invention also provides a host cell containing the gene encoding the GLP-1 or analogue recombinant tandem protein sequence or the recombinant plasmid. In some embodiments, the host cell is e.coli, preferably BL21(DE3), HMS174(DE3), pLysS or pLysE host bacteria.
In the second aspect of the invention, a method for preparing GLP-1 or an analogue thereof by using escherichia coli expression is provided, wherein the escherichia coli expression is adopted to express the recombinant tandem protein sequence of GLP-1 and the analogue thereof.
In a specific embodiment, the invention provides a method for preparing GLP-1 or an analogue thereof by using Escherichia coli expression.
In another preferred embodiment, the method for transforming the expression vector into E.coli is well known in the art, and electroporation, CaCl2 transformation, etc. are preferred; the method for screening positive clones is well known in the art, and a resistance gene method is preferred; the method for inducing the host to express the protein is a technique known to those skilled in the art. Preferably IPTG at a concentration of 0.lmM to 10mM, at a temperature of 25-37 ℃ so that more efficient expression of the protein of interest can be obtained.
In another preferred embodiment, the method for disrupting bacterial cells is preferably high-pressure homogenization, osmotic pressure shock, freeze thawing, or ultrasonic disruption.
In another preferred embodiment, the inclusion body is obtained by centrifugation.
The inclusion body formed by the GLP-1 and the analog recombinant tandem protein sequence thereof has good solubility, can completely dissolve the expressed inclusion body without a denaturant, and is very favorable for subsequent enzyme digestion and separation and purification. The invention provides a preferable method for dissolving inclusion bodies, which is to preferably dissolve 25-250mM Tris solution at the pH of 8.5-11.5 and at the temperature of 12-15 ℃ for 2-6 h.
In another preferred embodiment, the method for obtaining the crude GLP-1 or the analogue recombinant tandem protein lysate is preferably centrifugation or ultrafiltration.
In another preferred embodiment, the method for obtaining crude GLP-1 or its analogue monomer is preferably a trypsin and CPB enzyme double-enzyme cleavage method, and a Kex2 enzyme and CPB enzyme double-enzyme cleavage method.
In another preferred embodiment, the trypsin (or Kex2 enzyme) and CPB enzyme are used in a double enzyme cleavage method under the conditions commonly used for such enzyme cleavage, for example, the buffer may be Tris solution (25-250mM) or phosphate buffer; pH 7.0-10.0, preferably pH 8.0-9.0; the temperature is 15-30 deg.C, preferably 25 deg.C, and both enzymes are added according to the total protein mass in series of 1/400-1/800, preferably 1/500; the enzyme cutting time is 4-12 h. The HPLC purity of the target protein after enzyme digestion is not lower than 80%, which is very beneficial to the subsequent protein purification process. The enzyme cutting yield is not less than 45 percent and is 200 to 300 percent of the enzyme cutting yield of the traditional fusion protein.
An expression technology for designing a novel GLP-1 or a recombinant polypeptide tandem sequence of an analogue thereof by utilizing a genetic engineering means. The recombinant tandem protein can be produced by a prokaryotic expression system, GLP-1 or an analogue thereof with high yield can be obtained by enzyme digestion, and the product can be subjected to side chain modification and is used for treating diabetes and endocrine metabolism related diseases.
The invention designs an effective connecting sequence near the enzyme cutting site between the target genes expressed in series, and the fusion protein is replaced by GLP-1 or analog protein thereof to form series expression, the connecting sequence can fully expose the enzyme cutting site and completely cut off the connecting sequence and the recognition site, thereby obviously improving the enzyme cutting yield and purity of the target product, and leading the main protein after enzyme cutting to be the target protein of GLP-1 or analog thereof, thus greatly reducing the content of useless fusion protein, obviously improving the proportion of GLP-1 or analog protein thereof in the whole expression peptide chain, leading the theoretical yield after enzyme cutting to be close to 70 percent, and being a good method for effectively improving the expression yield of GLP-1 or analog protein thereof. Another advantage of the protein produced by this route is the good solubility of inclusion bodies. The protein product expressed by the traditional escherichia coli is mostly in a compact inclusion body form, the solubility of the inclusion body is poor, and the inclusion body is dissolved by using a denaturant such as high-concentration urea or guanidine hydrochloride.
Drawings
FIG. 1 shows an electrophoresis picture of the recombinant 3 tandem protein 1 engineering bacteria (SEQ ID No.30) induced expression of the somaglutide precursor. Wherein 1 is marker, 2 is post-induction engineering bacteria, and 2 is pre-induction engineering bacteria.
FIG. 2 shows an electrophoresis picture of the recombinant 3 tandem protein 2 engineering bacteria (SEQ ID No.38) induced expression of the somaglutide precursor. Wherein, 1 is engineering bacteria before induction, 2 is marker, and 3 is engineering bacteria after induction.
FIG. 3 shows the electrophoresis picture of the recombinant 4 tandem protein 1 engineering bacterium (SEQ ID No.92) induced expression of the somaglutide precursor. Wherein, 1 is engineering bacteria before induction, 2 is marker, and 3 is engineering bacteria after induction.
FIG. 4 shows an electrophoresis picture of the recombinant 4 tandem protein 2 engineering bacteria (SEQ ID No.109) induced expression of the somaglutide precursor. Wherein 1 is marker, 2 is engineering bacteria before induction, and 3 is engineering bacteria after induction.
FIG. 5 is a HPLC check chart of recombinant 3 tandem protein 1(SEQ ID No.30) of the Somalutide precursor after double cleavage by Kex2 and CPB. The somarglutide precursor peaked at RT6.349min and the purity was 85.06%.
FIG. 6 is a HPLC check chart of recombinant 3 tandem protein 2(SEQ ID No.38) of the Somalutide precursor after double cleavage by Kex2 and CPB. The somalobtide precursor peaked at rt6.323min with a purity of 86.27%.
FIG. 7 is a HPLC check chart of recombinant 4 tandem protein 1(SEQ ID No.92) of the Somalutide precursor after double cleavage by Kex2 and CPB. The somaglutide precursor peaks at RT6.334min and has a purity of 85.33%.
FIG. 8 is a HPLC check chart of recombinant 4 tandem protein 2(SEQ ID No.109) of the Somalutide precursor after double cleavage by Kex2 and CPB. The somalutide precursor peaked at rt6.261min with a purity of 83.89%.
FIG. 9 is a mass spectrum analysis diagram of recombinant 3 tandem protein 1(SEQ ID No.30) of the Somalutide precursor after double cleavage by Kex2 and CPB. The molecular weight of the somasu peptide is 3175, which is consistent with theory.
FIG. 10 is a mass spectrum analysis diagram of recombinant 3 tandem protein 2(SEQ ID No.38) of the Somalutide precursor after double cleavage by Kex2 and CPB. The molecular weight of the somasu peptide is 3175, which is consistent with theory.
FIG. 11 is a mass spectrum analysis diagram of recombinant 4 tandem protein 1(SEQ ID No.92) of the Somalutide precursor after double cleavage by Kex2 and CPB. The molecular weight of the somasu peptide is 3175, which is consistent with theory.
FIG. 12 is a mass spectrum analysis diagram of recombinant 4 tandem protein 2(SEQ ID No.109) of the Somalutide precursor after double cleavage by Kex2 and CPB. The molecular weight of the somasu peptide is 3175, which is consistent with theory.
Detailed Description
The present invention will be further described with reference to the following examples, which are intended to illustrate the present invention and not to limit the scope of the present invention, and all simple modifications of the preparation method of the present invention based on the idea of the present invention are within the scope of the present invention. The following examples are experimental methods without specifying specific conditions, and generally follow the methods known in the art.
Materials:
strains and plasmids: expression strains BL21(DE3), pLysS and the like were purchased from MERCK, and pET series vectors were purchased from vast Ling Biotech Co., Ltd.
Enzymes and reagents:
the enzymes used in the examples relating to molecular biological manipulations were purchased from Takara and the corresponding procedures were carried out exactly according to the relevant product instructions.
The gene synthesis and sequencing of the nucleotide sequence encoding the recombinant somalutide tandem proteins referred to in the examples was performed by the Nanjing Kinsley Limited.
Other raw materials and auxiliary materials which are not marked are all commercial products.
Example 1:
sequence design 1 of GLP-1 analogue triple tandem protein
In order to increase the expression amount of a GLP-1 analogue precursor peptide in prokaryotic expression and improve the proportion of a precursor peptide chain in the whole expression peptide chain, according to the above patent idea, the GLP-1 analogue precursor peptide chain is subjected to triple tandem design (n is 3), and GLP-1 analogue precursors are connected through a trypsin enzyme cutting site or a kex2 enzyme cutting site, so that trypsin or kex2 enzyme can better recognize the trypsin enzyme cutting site or the kex2 enzyme cutting site and the enzyme cutting efficiency is improved, and a key auxiliary sequence (m) is designed at the trypsin enzyme cutting site or the kex2 enzyme cutting site. GLP-1 analogues (somaglutide precursor sequence (9-37) and liraglutide precursor sequence (7-37)) published according to NCBI have the leader peptide MRLNSA at its N-terminus, with kex2 enzymatic cleavage sites and helper sequences incorporated between the GLP-1 native sequence and the leader peptide and between the GLP-1 native sequences: GSGSEEGSGS or GSGSDDGSGS or GSGSEDGSGS or GSGSDEGSGS or EEAE or DDAD or TEEAEKL or TDDADKL or TEDAEKL or TDEADKL or EGTEEAEKLG or EGTDDADKLG or EGTEEADKLG or EGTDDAEKLG or EGTEDADKLG or EGTDEAEKLG, and n is 3.
Therefore, the sequences of the designed GLP-1 natural analogues (the somaglutide precursor sequence (79-37) and the liraglutide precursor sequence (7-37)) recombinant 3 tandem proteins are shown in Table 1:
[ TABLE 1 ]
Figure BDA0002340321340000061
Figure BDA0002340321340000071
Figure BDA0002340321340000081
Figure BDA0002340321340000091
Sequence design 2 of GLP-1 analogue tetra-tandem protein
According to the above patent design concept, a GLP-1 analog precursor peptide chain is also designed in a quadruple tandem (n ═ 4) using a linker sequence consistent with the triple tandem design. GLP-1 analogues (somaglutide precursor sequence (9-37) and liraglutide precursor sequence (7-37)) published according to NCBI have the leader peptide MRLNSA at its N-terminus, with kex2 enzymatic cleavage sites and helper sequences incorporated between the GLP-1 native sequence and the leader peptide and between the GLP-1 native sequences: GSGSEEGSGS or GSGSDDGSGS or GSGSEDGSGS or GSGSDEGSGS or EEAE or DDAD or TEEAEKL or TDDADKL or TEDAEKL or TDEADKL or EGTEEAEKLG or EGTDDADKLG or EGTEEADKLG or EGTDDAEKLG or EGTEDADKLG or EGTDEAEKLG, and n is 4.
Therefore, the sequences of the designed GLP-1 natural analogues (the somaglutide precursor sequence (79-37) and the liraglutide precursor sequence (7-37)) recombinant 4 tandem proteins are shown in Table 2:
[ TABLE 2 ]
Figure BDA0002340321340000101
Figure BDA0002340321340000111
Figure BDA0002340321340000121
Figure BDA0002340321340000131
Figure BDA0002340321340000141
Example 2: construction of GLP-1 analogue tandem protein recombinant plasmid and construction of engineering bacteria
Nucleotide sequences corresponding to the designed amino acid sequences are optimized according to codons of escherichia coli respectively, the codon-optimized gene synthesis is carried out through a chemical synthesis method, and BamHI and XhoI are respectively connected into pET32a to construct 4 recombinant plasmids. Passing the above recombinant plasmid through CaC12The transformation method is introduced into BL21(DE3), and multiple strains of the somaglutide precursor recombinant 3 and 4 tandem protein engineering bacteria are constructed through resistance screening and monoclone. The sequence in the engineering bacteria is consistent with the design through sequencing.
Example 3: inducible expression of GLP-1 analogue tandem proteins
3.1 LB Medium formulation
TABLE 3 LB medium recipe
Figure BDA0002340321340000142
Preparing 11L LB medium according to Table 3, dissolving, mixing well, sucking 10ml and 1.7L medium, respectively placing into 50ml and 3L conical flask, preparing 6 bottles, sealing, and sterilizing (121 deg.C, 25min) for use.
3.2 engineering bacteria Resuscitation
One of the somalutide precursor recombinant 3 and 4 tandem protein glycerol strains was taken out, inoculated into 6 sterilized culture media of 10ml in an inoculum size of 1%, and cultured overnight in a shaker at 37 ℃ and 250 rpm.
3.3 Induction culture of engineering bacteria
Taking out the resuscitating bacteria liquid, respectively inoculating to 6 1.7L sterilized culture mediums according to the inoculation amount of 1%, culturing for about 7-8h under the conditions of 37 ℃ of a shaking table and 250rpm, adding 0.lmM IPTG, and carrying out overnight induction expression at 37 ℃.
3.4 centrifugation of the cells
Centrifuging the bacterial liquid at 4000rpm and 8 ℃ for 40min, removing supernatant and collecting thalli. Expression of the target protein in the cells was observed by SDS-PAGE (electrophoresis of the engineered bacteria with partially tandem proteins induced expression is shown in FIGS. 1-4).
Example 4: harvesting and solubilization of GLP-1 analog tandem protein inclusion bodies
4.1 resuspending, crushing and centrifuging the cells
Adding 10L of bacteria breaking buffer (5mmol/L EDTA, 50mmol/L Tris-HCl) into 1kg of bacteria to suspend the bacteria, fully stirring until no blocky bacteria exist, homogenizing the suspension at high pressure to break the bacteria, wherein the operating pressure adopted during the bacteria breaking is 850 +/-50 bar, and continuously breaking twice. The lysate liquid is centrifuged for 40min at 4000rpm and 8 ℃, and the precipitate is collected by centrifugation.
4.2 Inclusion body washing centrifugation
The final volume of the inclusion body wash was calculated according to the weight of inclusion bodies before washing according to the following formula: the final volume (L) of the inclusion body wash was defined as the weight (kg) of the inclusion body multiplied by the dilution factor (10) of the inclusion body, and 0.5L of 1mol/L of the ltis-HCl solution was added to each 10L of the inclusion body wash to prepare a buffer solution. Centrifuging at 6000rpm and 8 deg.C for 30min, and centrifuging to collect precipitate.
4.3 Inclusion body solubilization
Adding 12-15 ml of Tris solution (50mM) into every 1g of the inclusion bodies after washing and centrifugation, dissolving at low temperature (12-15 ℃), adjusting the pH to 11.5 by using NaOH solution after 0.5h, adjusting the pH to 11.5 by using NaOH solution for the second time after 0.5h, and then continuously dissolving for 2-6 h.
Example 5: obtaining of GLP-1 analogue tandem protein precursor
5.1 Ultrafiltration of Inclusion body solutions
After the dissolution of the inclusion bodies is finished, a centrifuge is used for centrifuging for 1h under the condition of 10000g to remove particles. The centrifuged solution was ultrafiltered using a 750K hollow fiber column, and the filtrate was collected by washing with 50mM Tris solution.
5.2 enzyme digestion
And (3) adjusting the pH value of the solution after ultrafiltration to 8.5-9.0 by using 6M HCl, adding 1g of Kex2 enzyme and 1g of CPB enzyme into every 500g of protein after filtration, and adding Kex2 enzyme and CPB enzyme for carrying out enzyme digestion reaction. Slowly stirring the enzyme digestion liquid to control the temperature of the enzyme digestion reaction to be 20-25 ℃, and performing the enzyme digestion reaction for 6-10 h. Samples were taken for HPLC and mass spectrometric detection (results for partial tandem proteins are shown in FIGS. 6-8 and 9-12), and the cleavage yield was calculated.
The results are shown in Table 4, which shows that the purity of the main peak ranges from 80% to 93% and the yield ranges from 45% to 60% by HPLC detection after Kex2 and CPB double enzyme digestion. The graph shows that the HPLC of the somalutide precursor detects that GLP-1 or the analogue thereof generates a peak at RT6.3min and the purity is about 85 percent. Mass spectrometry analysis: the somaglutide precursor has a molecular weight of about 3175, consistent with theory.
[ TABLE 4 ]
Figure BDA0002340321340000161
Figure BDA0002340321340000171
And (3) knotting: through the series design, the proportion of GLP-1 or analogues thereof in the whole expression band can be obviously improved, the enzyme digestion efficiency is improved to 40-60 percent, and the enzyme digestion yield is 200-300 percent of that of the fusion protein design scheme. Under the same fermentation conditions, the yield of the target protein of GLP-1 or the analogue thereof can be obviously improved. And the purity of the main peak (GLP-1 or the analogue thereof) after enzyme digestion reaches 80-93%, which is very beneficial to subsequent purification.
Sequence listing
<110> Ten thousand New medicine science and technology (Suzhou) Co., Ltd
Wanbang pharmaceutical science and technology Co Ltd of Jiangsu
<120> method for preparing GLP-1 or analog polypeptide thereof by adopting escherichia coli expression tandem sequence
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<211>6
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>10
Thr Glu Glu Ala Glu Lys
1 5
<210>11
<211>6
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>11
Thr Asp Asp Ala Asp Lys
1 5
<210>12
<211>6
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>12
Thr Glu Asp Ala Glu Lys
1 5
<210>13
<211>6
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>13
Thr Asp Glu Ala Asp Lys
1 5
<210>14
<211>10
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>14
Glu Gly Thr Glu Glu Ala Glu Lys Leu Gly
1 5 10
<210>15
<211>10
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>15
Glu Gly Thr Asp Asp Ala Asp Lys Leu Gly
1 5 10
<210>16
<211>10
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>16
Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly
1 5 10
<210>17
<211>10
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>17
Glu Gly Thr Asp Asp Ala Glu Lys Leu Gly
1 5 10
<210>18
<211>10
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>18
Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly
1 5 10
<210>19
<211>10
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>19
Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly
1 5 10
<210>20
<211>31
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>20
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly
20 25 30
<210>21
<211>31
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>21
His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
20 25 30
<210>22
<211>29
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>22
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
1 5 10 15
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
20 25
<210>23
<211>39
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>23
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>24
<211>39
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>24
His Ala Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Pro Ser
35
<210>25
<211>44
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>25
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Pro Pro Ser Lys Lys Lys Lys Lys Lys
35 40
<210>26
<211>30
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>26
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30
<210>27
<211>31
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>27
His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu
1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly
20 25 30
<210>28
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>28
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser
1 5 10 15
Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser
85 90 95
Gly Ser Glu Glu Gly Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>29
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>29
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Asp Asp Gly Ser
1 5 10 15
Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Gly Ser Gly Ser Asp Asp Gly Ser Gly Ser Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser
85 90 95
Gly Ser Asp Asp Gly Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>30
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>30
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Glu Asp Gly Ser
1 5 10 15
Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Gly Ser Gly Ser Glu Asp Gly Ser Gly Ser Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser
85 90 95
Gly Ser Glu Asp Gly Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>31
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>31
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser
1 5 10 15
Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser
85 90 95
Gly Ser Asp Glu Gly Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>32
<211>141
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>32
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser
1 5 10 15
Gly Ser Lys Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Lys
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Lys Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135 140
<210>33
<211>141
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>33
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser
1 5 10 15
Gly Ser Lys Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Lys
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Lys Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135 140
<210>34
<211>117
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>34
Met Arg Leu Asn Ser Ala Lys Arg Glu Glu Ala Glu Lys Arg Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Glu Ala
35 40 45
Glu Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
50 55 60
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
65 70 75 80
Lys Arg Glu Glu Ala Glu Lys Arg Glu Gly Thr Phe Thr Ser Asp Val
85 90 95
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
100 105 110
Val Arg Gly Arg Gly
115
<210>35
<211>117
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>35
Met Arg Leu Asn Ser Ala Lys Arg Asp Asp Ala Asp Lys Arg Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Asp Asp Ala
35 40 45
Asp Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
50 55 60
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
65 70 75 80
Lys Arg Asp Asp Ala Asp Lys Arg Glu Gly Thr Phe Thr Ser Asp Val
85 90 95
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
100 105 110
Val Arg Gly Arg Gly
115
<210>36
<211>126
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>36
Met Arg Leu Asn Ser Ala Lys ArgThr Glu Glu Ala Glu Lys Leu Lys
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
35 40 45
Thr Glu Glu Ala Glu Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Lys Arg Thr Glu Glu Ala Glu Lys Leu Lys
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
115 120 125
<210>37
<211>126
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>37
Met Arg Leu Asn Ser Ala Lys Arg Thr Asp Asp Ala Asp Lys Leu Lys
1 510 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
35 40 45
Thr Asp Asp Ala Asp Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Lys Arg Thr Asp Asp Ala Asp Lys Leu Lys
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
115 120 125
<210>38
<211>126
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>38
Met Arg Leu Asn Ser Ala Lys Arg Thr Glu Asp Ala Glu Lys Leu Lys
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val SerSer Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
35 40 45
Thr Glu Asp Ala Glu Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Lys Arg Thr Glu Asp Ala Glu Lys Leu Lys
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
115 120 125
<210>39
<211>126
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>39
Met Arg Leu Asn Ser Ala Lys Arg Thr Asp Glu Ala Asp Lys Leu Lys
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 2530
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
35 40 45
Thr Asp Glu Ala Asp Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Lys Arg Thr Asp Glu Ala Asp Lys Leu Lys
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
115 120 125
<210>40
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>40
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Glu Glu Ala Glu Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>41
<211>130
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>41
Met Arg Leu Asn Ser Ala Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu
1 5 10 15
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
20 25 30
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
35 40 45
Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
50 55 60
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
65 70 75 80
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Glu Glu Ala Glu
85 90 95
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
100 105 110
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
115 120 125
Arg Gly
130
<210>42
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>42
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Asp Asp Ala Asp Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu GlyGln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Asp Asp Ala Asp Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Asp Asp Ala Asp Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>43
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>43
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Glu Glu Ala Asp Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Glu Ala Asp Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>44
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>44
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Glu Glu Ala Asp Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
2025 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Glu Ala Asp Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>45
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>45
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Asp Asp Ala Glu Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Asp Asp Ala Glu Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Asp Asp Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>46
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>46
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Glu Asp Ala Asp Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp ValSer Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Asp Ala Asp Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>47
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>47
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Asp Glu Ala Glu Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Asp Glu Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>48
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>48
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser
1 5 10 15
Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser
85 90 95
Gly Ser Glu Glu Gly Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>49
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>49
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Asp Asp Gly Ser
1 510 15
Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
20 25 30
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
35 40 45
Arg Arg Gly Ser Gly Ser Asp Asp Gly Ser Gly Ser Arg Arg Glu Gly
50 55 60
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
65 70 75 80
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser Gly
85 90 95
Ser Asp Asp Gly Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp
100 105 110
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
115 120 125
Leu Val Arg Gly Arg Gly
130
<210>50
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>50
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Glu Asp Gly Ser
15 10 15
Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Gly Ser Gly Ser Glu Asp Gly Ser Gly Ser Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser
85 90 95
Gly Ser Glu Asp Gly Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>51
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>51
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser
1 5 10 15
Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser
85 90 95
Gly Ser Asp Glu Gly Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>52
<211>141
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>52
Met Arg Leu Asn Ser Ala Arg ArgGly Ser Gly Ser Glu Glu Gly Ser
1 5 10 15
Gly Ser Arg Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Arg
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Arg Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135 140
<210>53
<211>141
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>53
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser
1 5 10 15
Gly Ser Arg Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Arg
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Arg Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135 140
<210>54
<211>117
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>54
Met Arg Leu Asn Ser Ala Arg Arg Glu Glu Ala Glu Arg Arg Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Glu Ala
35 40 45
Glu Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
50 55 60
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
65 70 75 80
Arg Arg Glu Glu Ala Glu Arg Arg Glu Gly Thr Phe Thr Ser Asp Val
85 90 95
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
100 105 110
Val Arg Gly Arg Gly
115
<210>55
<211>117
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>55
Met Arg Leu Asn Ser Ala Arg Arg Asp Asp AlaAsp Arg Arg Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Asp Asp Ala
35 40 45
Asp Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
50 55 60
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
65 70 75 80
Arg Arg Asp Asp Ala Asp Arg Arg Glu Gly Thr Phe Thr Ser Asp Val
85 90 95
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
100 105 110
Val Arg Gly Arg Gly
115
<210>56
<211>126
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>56
Met Arg Leu Asn Ser Ala Arg Arg Thr Glu Glu Ala Glu Lys Leu Arg
1 5 10 15
Arg Glu Gly Thr PheThr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
35 40 45
Thr Glu Glu Ala Glu Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Arg Arg Thr Glu Glu Ala Glu Lys Leu Arg
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
115 120 125
<210>57
<211>126
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>57
Met Arg Leu Asn Ser Ala Arg Arg Thr Asp Asp Ala Asp Lys Leu Arg
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
2025 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
35 40 45
Thr Asp Asp Ala Asp Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Arg Arg Thr Asp Asp Ala Asp Lys Leu Arg
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
115 120 125
<210>58
<211>126
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>58
Met Arg Leu Asn Ser Ala Arg Arg Thr Glu Asp Ala Glu Lys Leu Arg
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala TrpLeu Val Arg Gly Arg Gly Arg Arg
35 40 45
Thr Glu Asp Ala Glu Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Arg Arg Thr Glu Asp Ala Glu Lys Leu Arg
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
115 120 125
<210>59
<211>126
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>59
Met Arg Leu Asn Ser Ala Arg Arg Thr Asp Glu Ala Asp Lys Leu Arg
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
35 4045
Thr Asp Glu Ala Asp Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Arg Arg Thr Asp Glu Ala Asp Lys Leu Arg
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
115 120 125
<210>60
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>60
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Glu Ala Glu Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Glu Glu Ala Glu Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>61
<211>130
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>61
Met Arg Leu Asn Ser Ala Glu Glu Ala Glu Lys Leu Gly Arg Arg Glu
1 5 10 15
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
20 25 30
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
35 40 45
Thr Glu Glu Ala Glu Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
50 55 60
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
65 70 75 80
Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly Thr Glu Glu Ala Glu
85 90 95
Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
100 105 110
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
115 120 125
Arg Gly
130
<210>62
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>62
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Asp Asp Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
GlyArg Arg Glu Gly Thr Asp Asp Ala Asp Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Asp Asp Ala Asp Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>63
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>63
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Glu Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Glu Glu Ala Asp Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>64
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>64
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Glu Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
3540 45
Gly Arg Arg Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Glu Glu Ala Asp Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>65
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>65
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Asp Asp Ala Glu Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Asp Asp Ala Glu Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Asp Asp Ala Glu Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>66
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>66
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile AlaTrp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Glu Asp Ala Asp Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>67
<211>135
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>67
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Asp Glu Ala Glu Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly
130 135
<210>68
<211>141
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>68
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly Arg
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly Arg Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135 140
<210>69
<211>141
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>69
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys
1 5 10 15
Leu Gly Arg Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly Arg
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly Arg Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
130 135 140
<210>70
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>70
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser
1 5 10 15
Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser
85 90 95
Gly Ser Glu Glu Gly Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser Gly Ser Glu Glu Gly
130 135 140
Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>71
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>71
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Asp Asp Gly Ser
1 5 10 15
Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Gly Ser Gly Ser Asp Asp Gly Ser Gly Ser Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser
85 90 95
Gly Ser Asp Asp Gly Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser Gly SerAsp Asp Gly
130 135 140
Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>72
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>72
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Glu Asp Gly Ser
1 5 10 15
Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Gly Ser Gly Ser Glu Asp Gly Ser Gly Ser Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser
85 90 95
Gly Ser Glu Asp Gly Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser Gly Ser Glu Asp Gly
130 135 140
Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>73
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>73
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser
1 5 10 15
Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
3540 45
Gly Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser
85 90 95
Gly Ser Asp Glu Gly Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Gly Ser Gly Ser Asp Glu Gly
130 135 140
Ser Gly Ser Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>74
<211>186
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>74
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser
1 5 10 15
Gly Ser Lys Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Lys
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Lys Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Lys Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly
130 135 140
Ser Gly Ser Glu Glu Gly Ser Gly Ser Lys Arg His Ala Glu Gly Thr
145 150 155 160
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
165 170 175
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
180 185
<210>75
<211>186
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>75
Met Arg Leu Asn Ser Ala Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser
1 5 10 15
Gly Ser Lys Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Lys
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Lys Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Lys Arg His Ala
100105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Gly
130 135 140
Ser Gly Ser Asp Glu Gly Ser Gly Ser Lys Arg His Ala Glu Gly Thr
145 150 155 160
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
165 170 175
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
180 185
<210>76
<211>154
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>76
Met Arg Leu Asn Ser Ala Lys Arg Glu Glu Ala Glu Lys Arg Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Glu Ala
35 40 45
Glu Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr LeuGlu
50 55 60
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
65 70 75 80
Lys Arg Glu Glu Ala Glu Lys Arg Glu Gly Thr Phe Thr Ser Asp Val
85 90 95
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
100 105 110
Val Arg Gly Arg Gly Lys Arg Glu Glu Ala Glu Lys Arg Glu Gly Thr
115 120 125
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
130 135 140
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
145 150
<210>77
<211>154
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>77
Met Arg Leu Asn Ser Ala Lys Arg Asp Asp Ala Asp Lys Arg Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Asp Asp Ala
35 40 45
Asp Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
50 55 60
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
65 70 75 80
Lys Arg Asp Asp Ala Asp Lys Arg Glu Gly Thr Phe Thr Ser Asp Val
85 90 95
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
100 105 110
Val Arg Gly Arg Gly Lys Arg Asp Asp Ala Asp Lys Arg Glu Gly Thr
115 120 125
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
130 135 140
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
145 150
<210>78
<211>166
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>78
Met Arg Leu Asn Ser Ala Lys Arg Thr Glu Glu Ala Glu Lys Leu Lys
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
35 40 45
Thr Glu Glu Ala Glu Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Lys Arg Thr Glu Glu Ala Glu Lys Leu Lys
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
115 120 125
Thr Glu Glu Ala Glu Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
130 135 140
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
145 150 155 160
Leu Val Arg Gly Arg Gly
165
<210>79
<211>166
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>79
Met Arg Leu Asn Ser Ala Lys Arg Thr Asp Asp Ala Asp Lys Leu Lys
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
35 40 45
Thr Asp Asp Ala Asp Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Lys Arg Thr Asp Asp Ala Asp Lys Leu Lys
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
115 120 125
Thr Asp Asp Ala Asp Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
130 135 140
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
145 150 155 160
Leu Val Arg Gly Arg Gly
165
<210>80
<211>166
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>80
Met Arg Leu Asn Ser Ala Lys Arg Thr Glu Asp Ala Glu Lys Leu Lys
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
35 40 45
Thr Glu Asp Ala Glu Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Lys Arg Thr Glu Asp Ala Glu Lys Leu Lys
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
115 120 125
Thr Glu Asp Ala Glu Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
130 135 140
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
145 150 155 160
Leu Val Arg Gly Arg Gly
165
<210>81
<211>166
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>81
Met Arg Leu Asn Ser Ala Lys Arg Thr Asp Glu Ala Asp Lys Leu Lys
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
35 40 45
Thr Asp Glu Ala Asp Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
6570 75 80
Leu Val Arg Gly Arg Gly Lys Arg Thr Asp Glu Ala Asp Lys Leu Lys
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg
115 120 125
Thr Asp Glu Ala Asp Lys Leu Lys Arg Glu Gly Thr Phe Thr Ser Asp
130 135 140
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
145 150 155 160
Leu Val Arg Gly Arg Gly
165
<210>82
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>82
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Glu Glu Ala Glu Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Glu Glu Ala Glu
130 135 140
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>83
<211>173
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>83
Met Arg Leu Asn Ser Ala Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu
1 5 10 15
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
20 25 30
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
35 40 45
Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
50 55 60
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
65 70 75 80
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Glu Glu Ala Glu
85 90 95
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
100 105 110
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
115 120 125
Arg Gly Lys Arg Glu Gly Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg
130 135 140
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
145 150155 160
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
165 170
<210>84
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>84
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Asp Asp Ala Asp Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Asp Asp Ala Asp Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Asp Asp Ala Asp Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala LysGlu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Asp Asp Ala Asp
130 135 140
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>85
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>85
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Glu Glu Ala Asp Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Glu Ala Asp Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Glu Glu Ala Asp
130 135 140
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>86
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>86
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Glu Glu Ala Asp Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
2025 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Glu Ala Asp Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Glu Glu Ala Asp
130 135 140
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>87
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>87
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Asp Asp Ala Glu Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Asp Asp Ala Glu Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Asp Asp Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Asp Asp Ala Glu
130 135 140
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>88
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>88
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Glu Asp Ala Asp Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Asp Ala Asp Lys Leu GlyLys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Glu Asp Ala Asp
130 135 140
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>89
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>89
Met Arg Leu Asn Ser Ala Lys Arg Glu Gly Thr Asp Glu Ala Glu Lys
1 5 10 15
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Lys Arg Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly Lys Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Asp Glu Ala Glu Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Asp Glu Ala Glu
130 135 140
Lys Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>90
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>90
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser
15 10 15
Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser
85 90 95
Gly Ser Glu Glu Gly Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser Gly Ser Glu Glu Gly
130 135 140
Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165170 175
Arg Gly
<210>91
<211>177
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>91
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Asp Asp Gly Ser
1 5 10 15
Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
20 25 30
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
35 40 45
Arg Arg Gly Ser Gly Ser Asp Asp Gly Ser Gly Ser Arg Arg Glu Gly
50 55 60
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
65 70 75 80
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser Gly
85 90 95
Ser Asp Asp Gly Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp
100 105 110
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
115 120 125
Leu Val Arg Gly Arg Gly Arg Arg Gly Ser Gly Ser Asp Asp Gly Ser
130 135 140
Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
145 150 155 160
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
165 170 175
Gly
<210>92
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>92
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Glu Asp Gly Ser
1 5 10 15
Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Gly Ser Gly Ser Glu Asp Gly Ser Gly Ser Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser
85 90 95
Gly Ser Glu Asp Gly Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser Gly Ser Glu Asp Gly
130 135 140
Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>93
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>93
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser
1 5 10 15
Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg GlyArg
35 40 45
Gly Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser
85 90 95
Gly Ser Asp Glu Gly Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Gly Ser Gly Ser Asp Glu Gly
130 135 140
Ser Gly Ser Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>94
<211>186
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>94
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser
1 5 10 15
Gly Ser Arg Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Arg
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Arg Arg Gly Ser Gly Ser Glu Glu Gly Ser Gly Ser Arg Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly
130 135 140
Ser Gly Ser Glu Glu Gly Ser Gly Ser Arg Arg His Ala Glu Gly Thr
145 150155 160
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
165 170 175
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
180 185
<210>95
<211>186
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>95
Met Arg Leu Asn Ser Ala Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser
1 5 10 15
Gly Ser Arg Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser Arg
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Arg Arg Gly Ser Gly Ser Asp Glu Gly Ser Gly Ser ArgArg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Gly
130 135 140
Ser Gly Ser Asp Glu Gly Ser Gly Ser Arg Arg His Ala Glu Gly Thr
145 150 155 160
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
165 170 175
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
180 185
<210>96
<211>154
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>96
Met Arg Leu Asn Ser Ala Arg Arg Glu Glu Ala Glu Arg Arg Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 25 30
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Glu Ala
35 40 45
Glu Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
50 55 60
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
65 70 75 80
Arg Arg Glu Glu Ala Glu Arg Arg Glu Gly Thr Phe Thr Ser Asp Val
85 90 95
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
100 105 110
Val Arg Gly Arg Gly Arg Arg Glu Glu Ala Glu Arg Arg Glu Gly Thr
115 120 125
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
130 135 140
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
145 150
<210>97
<211>154
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>97
Met Arg Leu Asn Ser Ala Arg Arg Asp Asp Ala Asp Arg Arg Glu Gly
1 5 10 15
Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys
20 2530
Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Asp Asp Ala
35 40 45
Asp Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
50 55 60
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
65 70 75 80
Arg Arg Asp Asp Ala Asp Arg Arg Glu Gly Thr Phe Thr Ser Asp Val
85 90 95
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
100 105 110
Val Arg Gly Arg Gly Arg Arg Asp Asp Ala Asp Arg Arg Glu Gly Thr
115 120 125
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
130 135 140
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
145 150
<210>98
<211>166
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>98
Met Arg Leu Asn Ser Ala Arg Arg Thr Glu Glu Ala Glu Lys Leu Arg
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
35 40 45
Thr Glu Glu Ala Glu Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Arg Arg Thr Glu Glu Ala Glu Lys Leu Arg
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
115 120 125
Thr Glu Glu Ala Glu Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
130 135 140
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
145 150 155 160
Leu Val Arg Gly Arg Gly
165
<210>99
<211>166
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>99
Met Arg Leu Asn Ser Ala Arg Arg Thr Asp Asp Ala Asp Lys Leu Arg
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
35 40 45
Thr Asp Asp Ala Asp Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Arg Arg Thr Asp Asp Ala Asp Lys Leu Arg
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
115 120 125
Thr Asp Asp Ala Asp Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
130 135 140
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
145 150 155 160
Leu Val Arg Gly Arg Gly
165
<210>100
<211>166
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>100
Met Arg Leu Asn Ser Ala Arg Arg Thr Glu Asp Ala Glu Lys Leu Arg
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
35 40 45
Thr Glu Asp Ala Glu Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Arg Arg Thr Glu Asp Ala Glu Lys Leu Arg
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
115 120 125
Thr Glu Asp Ala Glu Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
130 135 140
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
145 150 155 160
Leu Val Arg Gly Arg Gly
165
<210>101
<211>166
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>101
Met Arg Leu Asn Ser Ala Arg Arg Thr Asp Glu Ala Asp Lys Leu Arg
1 5 10 15
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
20 25 30
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
35 40 45
Thr Asp Glu Ala Asp Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
50 55 60
Val Ser Ser Tyr Leu Glu Gly GlnAla Ala Lys Glu Phe Ile Ala Trp
65 70 75 80
Leu Val Arg Gly Arg Gly Arg Arg Thr Asp Glu Ala Asp Lys Leu Arg
85 90 95
Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
100 105 110
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg
115 120 125
Thr Asp Glu Ala Asp Lys Leu Arg Arg Glu Gly Thr Phe Thr Ser Asp
130 135 140
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
145 150 155 160
Leu Val Arg Gly Arg Gly
165
<210>102
<211>177
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>102
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Glu Ala Glu Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Glu Glu Ala Glu Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys Arg Glu Gly
85 90 95
Thr Glu Glu Ala Glu Lys Leu Gly Lys Arg Gly Thr Phe Thr Ser Asp
100 105 110
Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
115 120 125
Leu Val Arg Gly Arg Gly Lys Arg Glu Gly Thr Glu Glu Ala Glu Lys
130 135 140
Leu Gly Lys Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
145 150 155 160
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
165 170 175
Gly
<210>103
<211>173
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>103
Met Arg Leu Asn Ser Ala Glu Glu Ala Glu Lys Leu Gly Arg Arg Glu
1 5 10 15
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
20 25 30
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
35 40 45
Thr Glu Glu Ala Glu Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
50 55 60
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
65 70 75 80
Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly Thr Glu Glu Ala Glu
85 90 95
Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
100 105 110
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
115 120 125
Arg Gly Arg Arg Glu Gly Thr Glu Glu Ala Glu Lys Leu Gly Arg Arg
130 135 140
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu GlyGln Ala
145 150 155 160
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
165 170
<210>104
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>104
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Asp Asp Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Asp Asp Ala Asp Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Asp Asp Ala Asp Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly Thr Asp Asp Ala Asp
130 135 140
Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>105
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>105
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Glu Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Glu Glu Ala Asp Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly Thr Glu Glu Ala Asp
130 135 140
Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>106
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>106
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Glu Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Glu Glu Ala Asp Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Glu Glu Ala Asp Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly Thr Glu Glu Ala Asp
130 135 140
Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>107
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>107
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Asp Asp Ala Glu Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Asp Asp Ala Glu Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Asp Asp Ala Glu Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly Thr Asp Asp Ala Glu
130 135 140
Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>108
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>108
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 9095
Thr Glu Asp Ala Asp Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly Thr Glu Asp Ala Asp
130 135 140
Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>109
<211>178
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>109
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys
1 5 10 15
Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
20 25 30
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg
35 40 45
Gly Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly Arg Arg Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly
85 90 95
Thr Asp Glu Ala Glu Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser
100 105 110
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala
115 120 125
Trp Leu Val Arg Gly Arg Gly Arg Arg Glu Gly Thr Asp Glu Ala Glu
130 135 140
Lys Leu Gly Arg Arg Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
145 150 155 160
Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly
165 170 175
Arg Gly
<210>110
<211>186
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>110
Met Arg Leu Asn Ser AlaArg Arg Glu Gly Thr Glu Asp Ala Asp Lys
1 5 10 15
Leu Gly Arg Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly Arg
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Arg Arg Glu Gly Thr Glu Asp Ala Asp Lys Leu Gly Arg Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu
130 135 140
Gly Thr Glu Asp Ala Asp Lys Leu Gly Arg Arg His Ala Glu Gly Thr
145 150 155 160
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
165 170 175
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
180 185
<210>111
<211>186
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>111
Met Arg Leu Asn Ser Ala Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys
1 5 10 15
Leu Gly Arg Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
20 25 30
Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg
35 40 45
Gly Arg Gly Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly Arg
50 55 60
Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
65 70 75 80
Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
85 90 95
Arg Arg Glu Gly Thr Asp Glu Ala Glu Lys Leu Gly Arg Arg His Ala
100 105 110
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala
115 120 125
Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Arg Arg Glu
130 135 140
Gly Thr Asp Glu Ala Glu Lys Leu Gly Arg Arg His Ala Glu Gly Thr
145 150 155 160
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu
165 170 175
Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
180 185

Claims (10)

1. A GLP-1 or an analogue thereof recombinant tandem protein sequence is characterized in that the general formula of the amino acid sequence is as follows: x-
(Y-Z) n; wherein:
x is a leader peptide formed from 5-10 amino acids, the amino acid composition including but not limited to: alanine, leucine, asparagine, arginine, serine, phenylalanine, histidine, methionine, valine, phenylalanine, methionine, valine, methionine, or valine; preferred sequence compositions are MMRLN or MMRLNSA or MMRLNSAFV;
y is a trypsin and CPB enzyme double-enzyme cutting site or a Kex2 enzyme and CPB enzyme double-enzyme cutting site and an auxiliary sequence, and the general formula is Y-m-Y; wherein y is R or K or RR or KK or RK or KR; m is a special connecting sequence formed by 4-12 amino acids; preferred sequence compositions are GSGSEEGSGS or GSGSDDGSGS or GSGSEDGSGS or GSGSDEGSGS or EEAE or DDAD or TEEAEKL or TDDADKL or TEDAEKL or TDEADKL or EGTEEAEKLG or EGTDDADKLG or EGTEEADKLG or EGTDDAEKLG or EGTEDADKLG or EGTDEAEKLG;
z is GLP-1 or an analogue thereof, and the amino acid sequence is selected from any one of SEQ ID No. 20-SEQ ID No. 27:
SEQ ID No.20：HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG、
SEQ ID No.21：HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG、
SEQ ID No.22：EGTFTSDVSSYLEGQAAKEFIAWLVRGRG、
SEQ ID No.23：HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS、
SEQ ID No.24：HAEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS、
SEQ ID No.25：HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK、
SEQ ID No.26：HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR、
SEQ ID No.27：HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG；
n is an integer from 2 to 7, preferably 3, 4 or 5.
2. The recombinant tandem protein sequence of GLP-1 or its analog according to claim 1, wherein the amino acid sequence is as shown in any one of SEQ ID No.28-SEQ ID No. 111.
3. A gene encoding the recombinant tandem protein sequence of GLP-1 or an analog thereof according to claim 1.
4. A recombinant plasmid or host cell comprising the gene of claim 3.
5. A method for preparing GLP-1 or an analogue thereof by using Escherichia coli expression, which is characterized in that the GLP-1 or the analogue thereof recombinant tandem protein sequence of claim 1 is expressed by using Escherichia coli.
6. The process as claimed in claim 4, using E.coli which is the host bacterium BL21(DE3), HMS174(DE3), pLysS or pLysE.
7. The method of claim 4, further comprising disrupting the biomass to obtain inclusion bodies, lysing the inclusion bodies, and performing enzymatic cleavage to obtain GLP-1 or an analog thereof.
8. The method of claim 4, wherein the inclusion body is solubilized by 25-250mM Tris solution at pH8.5-11.5 at 12-15 ℃ for 2-6 h.
9. The method of claim 4, wherein the enzyme digestion to obtain GLP-1 or its analog is trypsin and CPB enzyme double-enzyme digestion, or Kex2 enzyme and CPB enzyme double-enzyme digestion.
10. The process of claim 8, wherein the buffer used for the cleavage is a 25-250mM Tris solution or phosphate buffer, pH 7.0-10.0, preferably pH 8.0-9.0; the temperature is 15-30 deg.C, preferably 25 deg.C, and both enzymes are added according to the total protein mass in series of 1/400-1/800, and enzyme digestion is carried out for 4-12 h.
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111718407A (en) * 2020-08-14 2020-09-29 北京质肽生物医药科技有限公司 Purification method of glucagon-like peptide-1 analogue
CN113416683A (en) * 2021-06-01 2021-09-21 南昌大学 Escherichia coli Nissle1917 genetically engineered bacterium and preparation method and application thereof
CN113502310A (en) * 2021-09-10 2021-10-15 北京惠之衡生物科技有限公司 Method for preparing semaglutide precursor through high-density fermentation
WO2021249564A1 (en) * 2020-06-11 2021-12-16 宁波鲲鹏生物科技有限公司 Semaglutide derivative, and preparation method therefor and application thereof
CN113801234A (en) * 2020-06-11 2021-12-17 宁波鲲鹏生物科技有限公司 Somaltulipide derivative and application thereof
WO2023280133A1 (en) * 2021-07-06 2023-01-12 苏州康宁杰瑞生物科技有限公司 Fusion protein and application thereof
CN115850385A (en) * 2022-07-04 2023-03-28 北京惠之衡生物科技有限公司 Expression promoting peptide and application thereof
WO2023045996A1 (en) * 2021-09-26 2023-03-30 康霖生物科技（杭州）有限公司 Nucleic acid construct for gene therapy of carbohydrate metabolism-related diseases
CN116948013A (en) * 2023-04-28 2023-10-27 江苏创健医疗科技股份有限公司 Recombinant micromolecular collagen and expression system and preparation method thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4929553A (en) * 1987-05-29 1990-05-29 Canadian Patents & Development Ltd. Protease for specific processing of secreted proteins
CN101172996A (en) * 2006-09-29 2008-05-07 上海新生源医药研究有限公司 Connecting peptide and polypeptide amalgamation representation method for polypeptide amalgamation representation
CN110128552A (en) * 2019-03-14 2019-08-16 美药星（南京）制药有限公司 A kind of fusion protein and its method for preparing Liraglutide intermediate polypeptide
CN110305223A (en) * 2019-06-26 2019-10-08 重庆派金生物科技有限公司 The method that recombination fused in tandem albumen prepares target polypeptides

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4929553A (en) * 1987-05-29 1990-05-29 Canadian Patents & Development Ltd. Protease for specific processing of secreted proteins
CN101172996A (en) * 2006-09-29 2008-05-07 上海新生源医药研究有限公司 Connecting peptide and polypeptide amalgamation representation method for polypeptide amalgamation representation
CN110128552A (en) * 2019-03-14 2019-08-16 美药星（南京）制药有限公司 A kind of fusion protein and its method for preparing Liraglutide intermediate polypeptide
CN110305223A (en) * 2019-06-26 2019-10-08 重庆派金生物科技有限公司 The method that recombination fused in tandem albumen prepares target polypeptides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JIANHUA HOU,ET AL.: "High-Level Expression of Fusion Protein Containing 10 Tandem Repeated GLP-1 Analogs in Yeast Pichia pastoris and Its Biological Activity in a Diabetic Rat Model", 《BIOSCI.BIOTECHNOL.BIOCHEM.》 *
李泰明等: "重组人胰高血糖素样肽-1类似物的制备及活性分析", 《中国药科大学学报》 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021249564A1 (en) * 2020-06-11 2021-12-16 宁波鲲鹏生物科技有限公司 Semaglutide derivative, and preparation method therefor and application thereof
CN113801234A (en) * 2020-06-11 2021-12-17 宁波鲲鹏生物科技有限公司 Somaltulipide derivative and application thereof
CN113801234B (en) * 2020-06-11 2024-03-01 宁波鲲鹏生物科技有限公司 Sodamide derivative and application thereof
CN111718407A (en) * 2020-08-14 2020-09-29 北京质肽生物医药科技有限公司 Purification method of glucagon-like peptide-1 analogue
CN113416683A (en) * 2021-06-01 2021-09-21 南昌大学 Escherichia coli Nissle1917 genetically engineered bacterium and preparation method and application thereof
WO2023280133A1 (en) * 2021-07-06 2023-01-12 苏州康宁杰瑞生物科技有限公司 Fusion protein and application thereof
CN113502310A (en) * 2021-09-10 2021-10-15 北京惠之衡生物科技有限公司 Method for preparing semaglutide precursor through high-density fermentation
WO2023045996A1 (en) * 2021-09-26 2023-03-30 康霖生物科技（杭州）有限公司 Nucleic acid construct for gene therapy of carbohydrate metabolism-related diseases
CN115850385A (en) * 2022-07-04 2023-03-28 北京惠之衡生物科技有限公司 Expression promoting peptide and application thereof
CN115850385B (en) * 2022-07-04 2023-08-11 北京惠之衡生物科技有限公司 Expression-promoting peptide and application thereof
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