CN112759653A - PKEK fusion protein and preparation method and application thereof - Google Patents

PKEK fusion protein and preparation method and application thereof Download PDF

Info

Publication number
CN112759653A
CN112759653A CN201911075149.5A CN201911075149A CN112759653A CN 112759653 A CN112759653 A CN 112759653A CN 201911075149 A CN201911075149 A CN 201911075149A CN 112759653 A CN112759653 A CN 112759653A
Authority
CN
China
Prior art keywords
ala
lys
gly
glu
pro
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201911075149.5A
Other languages
Chinese (zh)
Other versions
CN112759653B (en
Inventor
刘合栋
曹春来
杨晓纯
杨祺
邹鑫
刘学明
梁雄基
王康钊
肖炳文
黄有泉
周翠
何秀仪
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhuhai United Laboratories Co Ltd
Original Assignee
Zhuhai United Laboratories Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhuhai United Laboratories Co Ltd filed Critical Zhuhai United Laboratories Co Ltd
Priority to CN201911075149.5A priority Critical patent/CN112759653B/en
Publication of CN112759653A publication Critical patent/CN112759653A/en
Application granted granted Critical
Publication of CN112759653B publication Critical patent/CN112759653B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Landscapes

  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a PKEK fusion protein and a preparation method and application thereof. The fusion protein is formed by connecting a plurality of flexible connecting peptides and PKEK in series in sequence; the protein is expressed in a form of inclusion body protein in microbial cells, and meanwhile, a plurality of flexible connecting peptides and the whitening polypeptide PKEK are sequentially connected in series to form fusion protein, so that miscut impurities generated in the enzyme digestion process can be effectively reduced, the high-purity whitening polypeptide PKEK can be efficiently obtained, and the purity can reach 95.4%. In addition, by using the preparation method for producing the whitening polypeptide PEKE by using the genetic engineering technology, a large amount of PKEK fusion protein inclusion bodies can be obtained within 12 hours, the expression amount can reach 41.5g/L at most, the process is simple and efficient, the amplification is easy, and the industrial production is facilitated.

Description

PKEK fusion protein and preparation method and application thereof
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a PKEK fusion protein, and a preparation method and application thereof.
Background
The most important functions of the skin are to protect the body from excessive water loss, and also to effectively prevent the entry of harmful chemicals or bacteria and solar radiation. If human skin is exposed to the environment of solar radiation for a long time, the skin responds to UV radiation through melanin formed, and finally photo-induced skin aging and pigment diseases are formed, outdoor ultraviolet UVB (280-320 nm) exists in the solar spectrum, and the problems that freckle, chloasma and the like are easily caused by excessive pigment deposition on the local part of the skin under the irradiation of the UVB. The pigmentation of the skin is essentially determined by the skin's own melanin, which is formed by specialized cells in the epidermis, i.e., melanocytes. During melanin synthesis, tyrosinase, as the starting enzyme, plays a decisive role; in keratinocytes, UV-A and UV-B radiation stimulate the formation of melanin in melanocytes by forming NO. Melanocytes respond to NO by increasing cell growth, forming a higher degree of branching and increasing melanin synthesis, while melanin production is controlled by the pigment deposition regulator (α -MSH). Various methods have been developed to reduce skin pigmentation, the most commonly used skin and hair lightening agent being hydroquinone or hydroquinone glycoside arbutin. However, these compounds have cytotoxic effects on melanocytes and irritant effects on the skin. A novel whitening polypeptide PKEK is described in patents W0/2008/08M94 and W0/2009/068351, and in-vitro cell experiments prove that the PKEK can effectively reduce the expression of interleukin-6, interleukin-8 and TNF-a and obviously reduce the expression of a pigmentation regulator (alpha-MSH) under the irradiation of ultraviolet UVB by human keratinocytes. In another random double-blind human skin efficacy test group, after 10 healthy subjects continuously use the PKEK once a day for four weeks, compared with a control group, the expression of interleukin-1 alpha, interleukin-6, interleukin-8, tumor necrosis factor-alpha and tyrosinase in the skin of a PKEK test group is obviously reduced under the irradiation of ultraviolet UVB; after the 39 caucasian women combined PKEK and vitamin C sodium phosphate for 6 weeks, facial spots obviously faded, but the spots faded obviously neither by PKEK nor vitamin C sodium phosphate alone; thus, both in vivo and in vitro experiments described above demonstrate that PKEK is effective in reducing facial pigmentation caused by outdoor uv light.
At present, the PKEK is mainly produced by a liquid phase synthesis method or a solid phase synthesis method (SPPS), because the side chains of lysine and glutamic acid which are used as raw materials for chemical synthesis are protected, a decapping process and crystallization are required after the transpeptidation reaction is finished, the operation is relatively complicated, and racemic chiral impurities are easily formed in the chemical synthesis process, so that the preparation cost of the high-purity PKEK is high. Therefore, the production of polypeptides by genetic engineering techniques is becoming a great trend.
Patent application CN107881187A discloses that EEEEK, EDK and DDDDK are used as connecting peptides, salt bonds are formed between side chain carboxyl of D or E and side chain amino of K to reduce acylation modification of K sites, however, the introduction of the connecting peptides formed by D or E in front of K does not affect the enzyme cutting efficiency of protease, and the problem of the miscut of PKEK in the subsequent enzyme cutting process cannot be solved.
Patent CN104725485A discloses a preparation method of recombinant active peptide, specifically, elastin-like purification tags ELPs and SUMO polypeptide are used for fusing one or more antihypertensive peptide monomers and antioxidant peptide monomers for expression, and target protein expressed by inclusion bodies is firstly denatured and renatured and then purified by tag technology; purifying fusion protein by ELPs label purification technology, adopting SUMO protease to crack, adding salt and centrifuging to remove diad fusion label ELPs-SUMO to obtain recombinant active peptide, and then adopting pepsin, trypsin and chymotrypsin to carry out enzymolysis on the recombinant active peptide and carrying out ultrafiltration to obtain a small peptide mixture. Although the method can prepare small peptides, the steps and the process are complicated, the SUMO protease with high cost and low enzyme digestion efficiency is needed, and the inclusion bodies need to be denatured and renatured before enzyme digestion. In addition, because SUMO proteases are not tolerant to denaturing solutions, it is necessary to remove the denaturing renaturation solutions by chromatographic techniques prior to enzymatic cleavage, resulting in the cost of small peptides prepared by biosynthetic methods not being a significant advantage over chemical synthesis.
Patent CN106560475A discloses a genetic engineering biosynthesis process of active short peptide, which comprises connecting active short peptide in series, expressing with purification tag in microbial cell as soluble protein or inclusion body protein, and degrading recombinant protein with high specificity protease to finally form single active short peptide. Although the method has certain advantages in the aspect of preparing single active short peptide, as the PKEK contains a plurality of basic amino acids, the isoelectric point of the plurality of PKEK is higher after the plurality of PKEK are connected in series, for example, the theoretical isoelectric point of 10 series-connected PKEK polypeptides is 9.92, the polypeptides containing a plurality of basic amino acids are usually expressed in a soluble protein form in microbial cells and are extremely difficult to form inclusion body protein, so that the polypeptides are easy to degrade, the lysine side chain amino group in the free PKEK series-connected polypeptides is easy to be acetylated and modified, and the method can not solve the problem of miscut of the PKEK series-connected polypeptides in the enzyme digestion process, so that the probability of finally obtaining the whitening polypeptide PKEK is greatly reduced.
Therefore, there is a need for an efficient method for producing the whitening polypeptide PKEK.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a PKEK fusion protein, wherein the fusion protein is expressed in a form of inclusion body protein in microbial cells, and meanwhile, a plurality of flexible connecting peptides and the whitening polypeptide PKEK are sequentially connected in series to form the fusion protein, so that miscut impurities generated in the enzyme digestion process can be effectively reduced, the high-purity whitening polypeptide PKEK is efficiently obtained, and the industrial production is facilitated.
A second object of the present application is to provide a gene encoding the above PKEK fusion protein.
A third object of the present application is to provide the use of the above-mentioned gene encoding a PKEK fusion protein.
The purpose of the invention is realized by the following technical scheme:
a PKEK fusion protein comprises a protein formed by sequentially connecting a plurality of flexible connecting peptides and a whitening polypeptide PKEK in series, and the structural formula of the protein is as follows: (B-PKEK)n(ii) a Wherein: b is flexible connecting peptide, and n is an integer of 1-30.
The PKEK fusion protein also comprises chaperonin, wherein the chaperonin is connected with a connecting peptide, and the structural formula is as follows: a- (B-PKEK)n(ii) a Wherein: a is chaperonin; b is flexible connecting peptide; n is an integer of 1 to 30; n is preferably 10-30; more preferably 10 to 20; more preferably 12 to 15.
The chaperone protein is KSI, PagP, PaP3.30, TAF12, mKSI, mPAgP, mPAP3.30 or mTAF12, and m represents a mutant. The chaperonin has the functions of assisting the fusion protein to form inclusion bodies in escherichia coli, promoting the formation of the inclusion bodies, improving the yield and quality of the inclusion bodies and reducing the risks of degradation and side chain modification; and facilitates downstream separation and purification. The existing wild-type aldosterone isomerase KSI fragment, wild-type PagP protein fragment, wild-type TAF12 protein fragment and wild-type PaP3.30 protein fragment all have high hydrophobicity, are easy to form inclusion bodies in escherichia coli, and can guide small peptides connected to the C end of the inclusion bodies to perform high-efficiency expression, so that KSI, PagP, PaP3.30 or TAF12 proteins are selected as chaperone proteins; in addition, because the wild-type chaperone protein has a plurality of lysine residues and arginine residues, excessive wrong enzyme cutting fragments can be formed in a subsequent enzyme cutting process, if lysine and arginine in the wild-type chaperone protein are mutated into other amino acids, the formation of impurities can be effectively reduced, and the enzyme cutting purity is obviously improved, so that the chaperone protein can also be a mutant of KSI, PagP, PaP3.30 or TAF12 protein, namely mKSI, mPAgP, mPAP3.30 or mTAF 12; preferably KSI, PagP or PaP3.30.
The flexible connecting peptide is Xm---X2-X1A peptide sequence as shown; wherein m is an integer of 0 to 10, preferably 1 to 7; x1Is a protease enzyme cutting site; x2To XmIs a flexible segment.
The protease restriction site is Arg or Lys; lys is preferred.
The amino acid in the flexible fragment cannot be Pro, Thr or Trp; preferably Gly, Ala or Ser.
The flexible connecting peptide is preferably AK, GK, AAK, AAGK, AAGGK, GGAAK, GGSASK or AAGGSGAGK.
When the chaperonin is KSI, the flexible connecting peptide is preferably AK, GK, AAK, AAGK, AAGGK, GGAAK or GGSASK; more preferably GK, AAK, AAGK, GGAAK or GGSASK; most preferably AAGK or GGAAK; the n is preferably 10-15; more preferably 12 to 15.
When the chaperonin is PagP, the flexible connecting peptide is preferably AK, GK, AAK, AAGK, AAGGK, GGAAK, GGSASK or AAGGSGAGK; more preferably AK, GK, AAKAAGK, AAGGK, GGAAK or GGSASK; the n is preferably 15-20.
When the chaperonin is PaP3.30, the flexible connecting peptide is preferably GGAAK and AAK; the n is preferably 10-30.
A gene encoding the above-mentioned PKEK fusion protein, obtainable by the codon rule; more preferably, the sequence is designed according to the codon usage preference of the host bacteria, such as the sequence shown in SEQ ID NO.37, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.56, SEQ ID NO.58, SEQ ID NO.60 and SEQ ID NO. 64.
A recombinant vector comprising a gene encoding the above PKEK fusion protein; the PKEK fusion protein is obtained by cloning the coding gene of the PKEK fusion protein into an expression vector.
The promoters on the expression vector comprise a T7 promoter, a T7Lac promoter, a Tac promoter, a Lac promoter and a Trp promoter; the T7 promoter and the T7lac promoter are preferred.
The expression vector is preferably a pET series vector; more preferably pET-3a, pET-9a, pET-28a (+), pET-22b (+), pET-26b (+), or pET-31b (+); most preferred are pET-31b (+) and pET28 a.
A recombinant expression strain containing the above recombinant vector; is obtained by transferring the recombinant vector into a genetic engineering strain.
The genetic engineering strain is selected from bacteria, yeast and fungi; preferably a bacterium; more preferred is Escherichia coli (Escherichia coli).
The PKEK fusion protein can be obtained by a chemical synthesis method or a genetic engineering method; preferably, the expression is obtained by a genetic engineering method, and the preparation method preferably comprises the following steps: fermenting and culturing the recombinant expression strain, and collecting inclusion bodies of the fusion protein to obtain the PKEK fusion protein.
The application of the PKEK fusion protein in the preparation of the whitening polypeptide PEKE specifically comprises the following steps: and dissolving and enzyme-cutting the inclusion body of the fusion protein, and separating and purifying to obtain the active whitening polypeptide PKEK.
A preparation method for producing whitening polypeptide PEKE by using a genetic engineering technology comprises the following steps:
s1, obtaining a recombinant vector containing the PKEK fusion protein coding gene, wherein the coded fusion protein is in the form of A- (B-PKEK)nWherein: a is chaperonin; b is flexible connecting peptide; n is 1 to 30An integer of (d);
s2, transferring the recombinant vector obtained in the step S1 into a genetic engineering strain to obtain a recombinant expression strain; fermenting and culturing the recombinant expression strain, and collecting inclusion bodies of the fusion protein;
s3, dissolving and enzyme cutting the inclusion body of the fusion protein, and separating and purifying to obtain the active whitening polypeptide PKEK.
The genetic engineering strain in the step S2 is bacteria, yeast or fungi, so that the recombinant expression vector containing the whitening polypeptide is introduced into the genetic engineering strain, new impurities are prevented from being introduced, and the subsequent industrial production is facilitated.
The bacterium is preferably Escherichia coli. The PKEK fusion protein can be expressed in an inclusion body form in escherichia coli, the fusion protein does not need renaturation, small molecular protein or polypeptide can be obtained only by dissolving the fusion protein under proper conditions and then directly diluting enzyme digestion, and the process is simple, easy to amplify and suitable for industrial production.
The fermentation medium used in the fermentation described in step S2 consists of an organic nitrogen source, a carbon source, and inorganic salts; specifically, the fermentation medium (total volume of 8L) was composed of the following components: 24g of yeast powder, 40g of peptone, 8.0g of NaCl and KH2PO4 24.0g、Na2HPO4 26.0g、MgSO4 8.0g、(NH4)2SO4 48.0g、CaCl2·2H20.11g of O, 33.0g of glycerol and 7mL of trace elements; after sterilization, the pH was adjusted to 7.0 with ammonia.
Wherein the mixed solution of the trace elements (total volume 100mL) comprises the following components: FeCl2·4H2O 2.287g、ZnCl2 0.131g、CoCl2·6H2O0.2g、Na2Mo4·2H2O 0.2g、CaCl2·2H2O 0.1g、CuCl2·2H2O 0.125g、H3BO4 0.05g、MnSO4·H2O0.217 g, and concentrated hydrochloric acid 10mL of 37 wt%.
The fermentation mode is high-density fermentation.
The inclusion body described in step S2 is obtained by disrupting the bacterial cells by a high-pressure homogenization method.
The protease used in the enzyme digestion in step S3 is any one or more of bovine trypsin, porcine trypsin, or lysyl-specific endonuclease.
The specific method for dissolving and enzyme-cutting the inclusion body of the fusion protein in the step S3 is as follows: dissolving the inclusion body of the collected fusion protein by using a high-concentration urea solution, diluting, and then carrying out enzyme digestion by using protease.
The concentration of the urea solution is 5M or more, more preferably 8M.
The dosage of the urea solution is preferably 1g of inclusion body: 5.5-6.5 mL of urea solution; more preferably 1g of inclusion bodies: 6mL of urea solution.
The preparation method for producing the whitening polypeptide PKEK by utilizing the genetic engineering technology is applied to the industrialized preparation of the whitening polypeptide PEKE. The preparation method for producing the whitening polypeptide PKEK by utilizing the genetic engineering technology is simple and efficient, and the prepared whitening polypeptide has high purity and is suitable for industrial production.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the inventors have discovered, inadvertently through creative work: when the carboxyl-terminal amino acid of the enzyme cutting site is a small side chain flexible amino acid, namely, the connecting peptide is connected behind the PKEK, the enzyme cutting efficiency is obviously improved. The inventor connects the flexible connecting peptide and the PKEK in series in sequence, so that the enzyme cutting efficiency of the second lysine in the PKEK is obviously higher than that of the first lysine, a large amount of complete PKEK polypeptide molecules are finally obtained, and miscut impurities PK and EK are obviously reduced; meanwhile, due to the introduction of the connecting peptide, part of the miscut impurities exist in the form of the connecting peptide + PK and EK + connecting peptide, so that the physicochemical properties of the miscut impurities and the complete PKEK whitening polypeptide are obviously different, the impurities can be effectively removed through a chromatography process, and the miscut impurities are controlled at a lower level.
The invention provides a PKEK fusion protein, which is expressed in a form of inclusion body protein in a microbial cell, and meanwhile, a plurality of flexible connecting peptides and a whitening polypeptide PKEK are sequentially connected in series to form the fusion protein, so that miscut impurities generated in the enzyme digestion process can be effectively reduced, and further the high-purity whitening polypeptide PKEK can be efficiently obtained, and the purity can reach 95.4%; in addition, by using the preparation method for producing the whitening polypeptide PEKE by using the genetic engineering technology, a large amount of PKEK fusion protein inclusion bodies can be obtained within 12 hours, the expression amount can reach 41.5g/L at most, the process is simple and efficient, the amplification is easy, and the industrial production is facilitated.
Drawings
FIG. 1 shows the recombinant plasmid pET31b-KSI- (AAK-PKEK) described in example 110Map of the plasmid.
FIG. 2 shows the genetically engineered strain BL21(DE)/pET31b-KSI- (AAK-PKEK) provided in example 110Performing shake flask fermentation and induction for 3h to obtain an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) chart of a sample; wherein, Lane M is protein marker, Lane 1 is negative control, and Lanes 2-7 represent different monoclonals.
FIG. 3 shows the genetically engineered strain BL21(DE)/pET31b-KSI- (AAK-PKEK) provided in example 110Performing shake flask fermentation induction for 15h to obtain an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) chart of a sample; wherein, Lane M is protein marker, Lane 8 is negative control, and Lanes 9-14 represent different monoclonals.
FIG. 4 shows the genetically engineered strain BL21(DE)/pET31b-KSI- (AAK-PKEK) provided in example 110SDS-PAGE pattern of fermentation test; among them, lanes 1 and 3 show the yield of the fusion protein obtained by induction for 0h, and lanes 2 and 4 show the yield of the fusion protein obtained by induction for 12 h.
FIG. 5 is a HPLC chromatogram of the pool after PEKE purification provided in example 1.
Fig. 6 is a graph showing the result of high resolution mass spectrometry analysis of the whitening polypeptide PKEK prepared in example 1.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Material
Restriction enzymes AlwNI and XhoI were purchased from TaKaRa; (AAK-PKEK)10The fusion gene sequence (shown as SEQ ID NO: 1) was synthesized by Invitrogen, Guangzhou; agarose gel recovery kits were purchased from TIANGEN; DNA Ligation Kit purchased from TaKaRaA driver; escherichia coli DH5 α was purchased from Life Technologies; the plasmid extraction kit is purchased from TIANGEN; identification of proteins containing (AAK-PKEK)10Cloning of gene fragments sequencing by Life Technologies; escherichia coli BL21(DE3) was purchased from Life Technologies; an improved Lowry method protein concentration determination kit is purchased from Shanghai Kogyo Co., Ltd in the field of biological engineering; the stirred fermenter was purchased from eastern Biometrics Ltd; the recombinant bovine trypsin is purchased from Shanghai Yaxin biotechnology, Inc.; recombinant porcine trypsin was purchased from Roche reagent; recombinant lysyl endonuclease was purchased from japan and wako pure chemical industries; uni PMM30-500 fillers are available from su nano technologies ltd.
Example 1
A preparation method for producing a whitening polypeptide PKEK by utilizing a genetic engineering technology comprises the following steps:
1. construction of recombinant plasmid pET31b-KSI- (AAK-PKEK)10
1.1 Artificial synthesis of a peptide linker and a PKEK gene sequence (AAK-PKEK)10
Design of linker peptide and PKEK Gene sequences (AAK-PKEK)10The fusion gene fragment of (1) converting the amino acid sequences into nucleotide sequences according to a codon table, selecting codons with higher use frequency according to codon use preference of escherichia coli in the conversion process, adjusting GC content of the codons, removing cis-acting elements and repetitive sequences influencing gene transcription to optimize the codons, introducing double-stop codons TAATGA at the 3' end of the gene sequence, and facilitating gene operation in (AAK-PKEK)10The 5' end of the fusion gene sequence is introduced with an AlwNI enzyme cutting site sequence CAGATGCTG, so that two ML amino acids are introduced before the N-terminal extension peptide (AAK-PKEK)10Introducing XhoI enzyme cutting site CTCGAG at the 3' end of the fusion gene, and optimizing (AAK-PKEK)10The fusion gene sequence is shown in SEQ ID NO. 1.
1.2pET31b-KSI-(AAK-PKEK)10Construction of recombinant expression vectors
Plasmid pET-31b (+) was double digested with restriction enzymes AlwNI and XhoI, and 5. mu.L of the digested product was applied to 0.8% agarose gelAfter the completion of the digestion was confirmed by electrophoresis, all the digested products were subjected to agarose gel electrophoresis at a mass/volume ratio of 0.8%, a gel containing a DNA fragment of about 5.7kb was excised, and the DNA fragment in the gel was purified to 30. mu.L of deionized water using an agarose gel recovery kit to obtain a plasmid fragment with a cohesive end. Similarly, the AlwNI and XhoI pair (AAK-PKEK)10And carrying out double enzyme digestion on the fusion gene fragment, and purifying the enzyme-digested DNA fragment into 20 mu L deionized water to obtain the gene fragment with the sticky end. The gene fragment and the plasmid fragment were ligated by using a DNA Ligation Kit (DNA Ligation Kit) under the conditions of 16 ℃ overnight, and 10. mu.L of the overnight ligated product was added to 80. mu.L of CaCl2 Escherichia coli DH 5. alpha. competent cells prepared by the method (third edition of molecular cloning Instructions published by Cold spring harbor laboratory in USA) were treated at 42 ℃ for 90s, 300. mu.L of LB liquid medium preheated at 37 ℃ was rapidly added (10 g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, pH 7.0-7.5), shaking-cultured in a shaker at low speed (100-150 rpm) at 37 ℃ for 45min, and 100. mu.L of the culture was spread with LB solid medium (10 g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, 15g/L of agar powder, pH 7.0-7.5) to which ampicillin was added (final concentration 100. mu.g/ml). Inversely culturing for about 18 hours in an incubator at 37 ℃ until a single colony grows out, randomly selecting a part of the single colony for carrying out bacteria liquid PCR identification, wherein the PCR reaction conditions are as follows: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 30 s; 5min at 72 ℃; the primers are as follows: a T7 promoter primer and a T7 terminator primer.
T7 promoter primer: 5'-TAATACGACTCACTATAGGG-3' (SEQ ID NO: 35);
t7 terminator primer: 5'-TGCTAGTTATTGCTCAGCGG-3' (SEQ ID NO: 36).
Extracting plasmid from the preliminarily screened positive clone by using a plasmid extraction kit according to the instruction, carrying out enzyme digestion identification by NdeI and XhoI to obtain a gene fragment of about 590bp and a vector fragment of about 5.3kb, namely the clone containing (AAK-PKEK)10Cloning of the gene fragment. Will identify the compound containing (AAK-PKEK)10The cloning of the gene fragment was sequenced. Sequencing was carried out to prove that there was no base mutation and no frame shift in readingThe single clone was inoculated into 50mL LB liquid medium supplemented with ampicillin (final concentration 100. mu.g/mL), cultured at 37 ℃ for 18h at 250rpm, and the resulting culture was plasmid-extracted using a plasmid extraction kit, named pET31b-KSI- (AAK-PKEK), as a recombinant expression vector10As shown in fig. 1.
2、BL21(DE3)/pET31b-KSI-(AAK-PKEK)10Screening of expression strains and product identification
Coli BL21(DE3) competent cells were prepared according to the calcium chloride method provided in the third edition of molecular cloning, A laboratory Manual, Cold spring harbor, USA. mu.L of the recombinant expression vector pET31b-KSI- (AAK-PKEK) of example 1 was taken10The above Escherichia coli competent cells were transformed by the calcium chloride method as described in 1.2. The transformation liquid was applied to LB solid medium supplemented with ampicillin (final concentration: 100. mu.g/mL), respectively, and inverted cultured at 37 ℃ until a single colony appeared, to obtain a strain bank expressing PKEK fusion protein and named BL21(DE3)/pET31b-KSI- (AAK-PKEK)10. Meanwhile, Escherichia coli BL21(DE3) competent cells were transformed with pET-31b (+) to give BL21(DE3)/pET31b strain as a negative control.
Respectively picking single colonies formed by the engineering bacteria in an LB solid culture medium by using a sterile toothpick, inoculating 3 single colonies into 50mL of LB liquid culture medium, carrying out shaking culture at 37 ℃ and 250rpm, inoculating 3-6 mL of bacterial liquid into 100mL of LB liquid culture medium when the OD600 of the bacterial liquid is 0.5-1.0, inoculating 3-6 mL of BL21(DE3)/pET31b bacterial liquid into 100mL of LB liquid culture medium as a negative control, adding IPTG (isopropyl-beta-D-thiogalactopyranoside) when carrying out shaking culture at 37 ℃ and 250rpm until the OD600 is 0.6-0.8, starting induction, respectively taking the bacterial liquid with the final concentration of 1mmol/L, carrying out centrifugation at 12000rpm for 1 minute, removing a supernatant, and preserving the bacterial bodies at-20 ℃ for later use.
The cells frozen at-20 ℃ were removed, 5mL of urea solution with a concentration of 8mol/L was added to resuspend the cells, and the cells were sonicated in an ice-water mixture for 10 minutes (sonication for 3 seconds, stop for 5 seconds, and so forth). Taking 15 mu L of the crushing liquid, adding 15 mu L of the loading buffer liquid, fully mixing uniformly, taking 10 mu L of the crushing liquid, carrying out SDS-PAGE (wherein the concentrated Gel contains 5 volume percent of acrylamide-methylene bisacrylamide (prepared according to the volume ratio of 29: 1) and the separation Gel contains 15 volume percent of acrylamide-methylene bisacrylamide. the electrophoresis conditions are that the current of the concentrated Gel is set to be 11mA and the current of the separation Gel is set to be 22 mA., taking out the Gel, staining the Gel with Coomassie brilliant blue staining solution (containing 0.6g of Coomassie brilliant blue R-250, 450mL of ethanol, 100mL of glacial acetic acid and the balance of purified water per liter) overnight, decoloring the Gel with decoloring solution (containing 250mL of ethanol, 80mL of glacial acetic acid and the balance of purified water per liter) until the background is transparent, photographing the Gel to extract an image under the transparent background, and analyzing the total strip accounting for the total strip by using a biological image processing software Gel-Pro Analyzer 4.0 as shown in figure 2 and figure 3 The ratio is the purity of the PKEK fusion protein in the total protein. And (3) operating by using an improved Lowry method protein concentration determination kit according to an instruction, determining the total protein concentration of the thallus crushing liquid, and converting by using a dilution multiple to obtain the total protein concentration of the bacteria liquid. And (4) converting the purity of the PKEK fusion protein in the total protein and the total protein concentration of the bacterial liquid to obtain the expression quantity of the PKEK fusion protein.
According to the electrophoresis result, the genetically engineered bacterium BL21(DE3)/pET31b-KSI- (AAK-PKEK)10The target protein is not expressed before induction, so that the toxicity of the target protein to host cells can be effectively reduced, and the protein expression time can be prolonged. The control bacterium BL21(DE3)/pET-31b (+) without PKEK gene can express aldosterone isomerase with molecular weight of about 14kDa after IPTG induction, while the gene engineering bacterium BL21(DE3)/pET31b-KSI- (AAK-PKEK)10The protein with the size of about 22kDa can be expressed after IPTG induction, which is consistent with the theoretical molecular weight of 21.2KDa (obtained by using online software http:// web. expasy. org/computer _ pi/prediction) of the PKEK fusion protein, and indicates that the PKEK gene has been successfully introduced into the genetic engineering bacteria.
The genetic engineering bacteria BL21(DE3)/pET31b-KSI- (AAK-PKEK)10Streaking on a fresh LB solid medium added with ampicillin with a final concentration of 100 mug/mL for pure culture, selecting a single colony, culturing the single colony with an LB liquid medium until OD600 is 0.8-1.0, preparing a bacterial solution containing 15% by volume of glycerol, storing the bacterial solution in a refrigerator at-70 ℃ for preparationThe application is as follows.
3. PKEK fusion protein gene engineering bacteria BL21(DE3)/pET31b-KSI- (AAK-PKEK)10Fermentation test
3.1 preparation of seed cultures
20 μ L of strain BL21(DE3)/pET31b-KSI- (AAK-PKEK) cryopreserved at-70 deg.C was taken10The culture was inoculated into 50mL of LB liquid medium supplemented with ampicillin (final concentration: 100. mu.g/mL), cultured on a shaker at 28 ℃ and 250rpm for 16 hours to activate the seed culture, 50mL of the activated seed culture was inoculated into 400mL of LB liquid medium, and further cultured at 28 ℃ and 250rpm for 3 hours to obtain a seed culture, and the OD600 of the cell concentration was controlled to be 2.0 to 3.0.
Fermentation culture in 3.220L fermenter
Using a 20L stirred tank fermenter, sterilizing according to the formula of the fermentation medium shown in Table 1, adjusting pH to 7.0 with ammonia water, feeding the material with a volume of 8L, and strictly controlling the fermentation conditions: the temperature is controlled to be 37 ℃, the pH is controlled to be 7.0, the Dissolved Oxygen (DO) is controlled to be between 5-50% of maximum oxygen saturation (the correction condition of the oxygen saturation is that saturated sodium sulfite solution is adopted, the correction condition of the maximum oxygen saturation is that the rotating speed is 150rpm, the air flow rate is 4L/min), the fermentation rotating speed is controlled to be between 150-700 rpm, the specific fermentation rotating speed is regulated and controlled according to the change of the DO, the air flow rate is controlled to be between 4-20L/min, feeding is started when the carbon source is cultured to be exhausted, wherein each kilogram of feeding liquid contains: 500g of glycerol, 33g of yeast extract powder, 66g of yeast peptone, 3.5mL of trace elements and the balance of water, wherein the total amount of the components is 0.4 mL-min-1·L-1The feeding rate of the feed is constant. When the cells were cultured to a cell concentration OD600 of about 80, IPTG addition was started to a final concentration of 0.3mM, and induction was started.
Table 1: fermentation medium formula
Components Dosage (g)
Yeast Extract (Yeast powder) 24.0
Tryptone (peptone) 40.0
NaCl 8.0
KH2PO4 24.0
Na2HPO4 26.0
MgSO4 8.0
(NH4)2SO4 48.0
CaCl2·2H2O 0.11
Glycerol 33.0
Microelement mixture (see Table 2) 7mL
Table 2: formula table of trace element mixed liquid (100mL)
Components Dosage (g)
FeCl2·4H2O 2.287
ZnCl2 0.131
CoCl2·6H2O 0.2
Na2Mo4·2H2O 0.2
CaCl2·2H2O 0.1
CuCl2·2H2O 0.125
H3BO4 0.05
MnSO4·H2O 0.217
37 wt% concentrated hydrochloric acid 10mL
Stopping fermentation after 12h of induction, taking 1mL of fermentation broth after 0h and 12h of induction, respectively, diluting with 10 times of purified water, and measuring (AAK-PKEK)10Expression of the fusion protein (two parallel batches).
Genetically engineered strain BL21(DE)/pET31b-KSI- (AAK-PKEK)10An SDS-PAGE pattern of a fermentation test is shown in FIG. 4, a liquid phase detection result of a collecting solution after PKEK purification is shown in FIG. 5, and the average yield of the inclusion bodies containing the fusion protein is 41.5g/L after conversion, wherein the content of the fusion protein in the inclusion bodies containing the fusion protein is 35 percent, namely 14.5 g/L.
4. Extraction and enzyme digestion of PKEK fusion protein inclusion body
4.1 isolation and extraction of Inclusion bodies
Centrifuging the fermentation liquor obtained in the step 3 at 4500rpm for 30 minutes to collect thalli, suspending the thalli in a crushing liquid (containing 6.05g of Tris and 1.46g of EDTA per liter and the balance of purified water and adjusting the pH to 8.0) according to the mass-to-volume ratio of 1:7, homogenizing and crushing the thalli under high pressure (900bar) in a water bath, centrifuging to collect precipitates, dissolving the collected precipitates in a washing buffer (containing 6.05g of Tris, 1.46g of EDTA, 2.9g of NaCl, 10mL of Tritiox-100 per liter and the balance of purified water) according to the mass-to-volume ratio of 1:15, washing, and centrifuging to collect the inclusion body precipitates.
4.2 Inclusion body denaturation liquid enzyme digestion
Dissolving 2.5g of the collected inclusion body with 15mL of denaturant (containing 8mol of urea per liter and the balance of purified water), diluting 1 time with 5mM Tris solution, adjusting the pH value to 9.0, and stirring at room temperature for 2-5 h.
And carrying out enzyme digestion by adopting recombinant bovine trypsin according to the specification, and obtaining a PKEK monomer crude product by enzyme digestion.
5. Purifying the enzyme-digested PKEK crude product
Loading 100mL of the crude enzyme-cleaved PKEK prepared in the above step 4 (fusion protein sequence is SEQ ID NO: 14) onto a chromatographic column filled with 10mL of Uni PMM30-500 filler (which is pre-equilibrated with a buffer solution containing 20% isopropanol containing 0.1% (v/v) TFA), after loading, washing with an equilibration buffer solution (a buffer solution containing 20% isopropanol containing 0.1% (v/v) TFA) until the baseline is stable, then eluting with a linear gradient of 100mL of 20% (v/v) isopropanol A solution (containing 0.1% (v/v) TFA buffer) and 100mL of 40% (v/v) isopropanol B solution (containing 0.1% (v/v) TFA buffer), wherein the elution flow rate is not more than 2 times the column volume per hour, collecting the main peak in segments, collecting about 20% of peak height, and the detection wavelength is 280nm, thus obtaining the PKEK prepared by the PKEK purification process as shown in FIG. 6, the purity reaches 95.4 percent.
PKEK liquid phase detection conditions: BEH-C8(1.7 μm particle size, 2.1X 100mm column) at 35 ℃ and a flow rate of 0.2 mL/min. Mobile phase A: aqueous solution containing 0.1% (v/v) TFA; mobile phase B: acetonitrile with 0.09% (v/v) TFA; gradient: the phase B is increased from 30% to 80% in 0-25 min; 25-38 min: phase B rose from 80% to 100%.
Mass spectrum identification: taking a PEKE main peak by a Seimerson high-resolution liquid chromatograph-mass spectrometer for high-resolution mass spectrometry, deconvoluting by using a Thermo Biopharma Finder2.0, and collecting the range: MS 100-1000. The result shows that the molecular weight of the PKEK prepared by the method is 500.3Da, as shown in FIG. 6, the molecular weight is completely consistent with the theoretical molecular weight, and the polypeptide prepared by the method is the high-purity whitening polypeptide PKEK.
Example 2
The method of this example is substantially the same as the method of example 1, except that the protease used for enzyme digestion in this example is recombinant porcine trypsin, the reaction is terminated by adjusting acid after reacting for 2 hours at 16 ℃, and the crude product of the PKEK monomer can be obtained by enzyme digestion.
The high-purity whitening polypeptide PKEK is efficiently prepared by the method.
Example 3
The method of this example is substantially the same as the method of example 1, except that the protease used for the enzyme digestion in this example is recombinant lysyl endonuclease, the reaction is terminated by adjusting acid after 2h at 25 ℃, and the crude product of the PKEK monomer can be obtained by the enzyme digestion.
Example 4
This example is essentially the same as example 1, except that the recombinant expression vector selected for use in this example contains different numbers of chaperones, linker peptides and PKEK concatemers. The relative yields of the different chaperone and linker fusion proteins are shown in table 3, and the relative purities of the modified enzyme-cleaved PKEK are shown in table 4.
Table 3: fermentation yield test of fusion proteins of different molecular chaperones and connecting peptides
Amino acid sequence Nucleic acid sequences Molecular chaperones Linker peptide Linker peptide-PKEK concatemeric Relative yield
SEQ ID NO:6 SEQ ID NO:1 Is free of AAK 10 Non-inclusion body
SEQ ID NO:7 SEQ ID NO:47 Is free of Is free of 10 Non-inclusion body
SEQ ID NO:8 SEQ ID NO:48 Is free of Is free of 20 Non-inclusion body
SEQ ID NO:9 SEQ ID NO:49 KSI Is free of 1 6％
SEQ ID NO:10 SEQ ID NO:50 KSI Is free of 10 42％
SEQ ID NO:11 SEQ ID NO:51 KSI AK 1 9％
SEQ ID NO:12 SEQ ID NO:52 KSI AK 10 91％
SEQ ID NO:13 SEQ ID NO:53 KSI GK 10 103％
SEQ ID NO:14 SEQ ID NO:40 KSI AAK 10 100％
SEQ ID NO:15 SEQ ID NO:54 KSI PPK 10 31％
SEQ ID NO:16 SEQ ID NO:41 KSI AAGK 12 120％
SEQ ID NO:17 SEQ ID NO:55 KSI AAGGK 10 98％
SEQ ID NO:18 SEQ ID NO:42 KSI GGAAK 15 132％
SEQ ID NO:19 SEQ ID NO:56 KSI GGSASK 10 101％
SEQ ID NO:20 SEQ ID NO:57 KSI AAGGSGAGK 10 89％
SEQ ID NO:21 SEQ ID NO:37 PagP AK 20 121％
SEQ ID NO:22 SEQ ID NO:58 PagP GK 20 145％
SEQ ID NO:23 SEQ ID NO:43 PagP AAK 20 140％
SEQ ID NO:24 SEQ ID NO:59 PagP WTK 20 55％
SEQ ID NO:25 SEQ ID NO:44 PagP AAGK 15 174％
SEQ ID NO:26 SEQ ID NO:45 PagP AAGGK 20 155％
SEQ ID NO:27 SEQ ID NO:46 PagP GGAAK 15 162％
SEQ ID NO:28 SEQ ID NO:60 PagP GGSASK 20 130％
SEQ ID NO:29 SEQ ID NO:61 PagP AAGGSGAGK 20 99％
SEQ ID NO:30 SEQ ID NO:38 TAF12 GGAAK 10 84％
SEQ ID NO:31 SEQ ID NO:62 TAF12 AAK 20 71％
SEQ ID NO:32 SEQ ID NO:39 PaP3.30 GGAAK 10 102％
SEQ ID NO:33 SEQ ID NO:63 PaP3.30 AAK 20 96％
SEQ ID NO:34 SEQ ID NO:64 PaP3.30 AAK 30 112％
Description of the drawings: the relative yields in the above table refer only to the relative yields of the PKEK moieties in the fusion protein molecules, from which the chaperone and linker peptide have been subtracted.
In the above table, the restriction sites of the recombinant vectors for protein molecules of SEQ ID NO. 9-20 are AlwNI and XhoI, and the restriction sites of the rest protein molecules are NdeI and XhoI. No connecting peptide exists in SEQ ID NO 7-10, so that Lys is inserted between a molecular chaperone and the first PKEK in order to ensure that complete PKEK can be obtained by subsequent enzyme digestion. The amino acid sequence of the KSI molecular chaperone is shown as SEQ ID NO. 2; the amino acid sequence of the PagP molecular chaperone is shown as SEQ ID NO. 3; the amino acid sequence of the molecular chaperone TAF12 is shown in SEQ ID NO. 4; the amino acid sequence of the PaP3.30 molecular chaperone is shown as SEQ ID NO. 5; the nucleotide sequence of the PagP molecular chaperone and connecting peptide fusion protein molecule (the amino acid sequence is shown as SEQ ID NO:21) is shown as SEQ ID NO: 37; the nucleotide sequences of the TAF12 molecular chaperone and the connecting peptide fusion protein molecule (the amino acid sequence is shown as SEQ ID NO:30) are shown as SEQ ID NO: 38; the nucleotide sequences of the molecular chaperone and the connecting peptide fusion protein molecule (the amino acid sequence is shown as SEQ ID NO:32) of the PaP3.30 are shown as SEQ ID NO: 39; the amino acid sequences of the different chaperone and linker peptide fusion protein molecules are shown as SEQ ID NO 6-34, and the nucleic acid sequences of the different chaperone and linker peptide fusion protein molecules are shown as SEQ ID NO 37-64, wherein the recombinant expression vector used in SEQ ID NO 14 was prepared in example 1 for comparative analysis. In the invention, the 5 'ends of all the nucleic acid sequences are enzyme cutting sites AlwNI or NdeI, the 3' ends are stop codons (taatga) and enzyme cutting sites XhoI, and all the amino acid sequences do not represent the enzyme cutting sites.
Table 4: the PKEK relative purity of the modified enzyme-digested PKEK of different molecular chaperones and connecting peptide fusion proteins
Figure BDA0002262207610000071
Figure BDA0002262207610000081
Description of the drawings: all groups in the table were digested with the same amount of PKEK in the inclusion bodies, with chaperones and linker peptides subtracted.
The results in Table 4 show that in the absence of linker peptide, the monomeric PKEK obtained by enzymatic cleavage is much lower than that obtained with linker peptide. While, when X in the peptide is linkedm---X2When the polypeptide is small side chain hydrophobic flexible polypeptide such as AA, GG, GGAA, GGSAS and the like, the PKEK monomer obtained after enzyme digestion is far higher than large side chain hydrophobic polypeptide such as WT, PP and the like. The large side chain hydrophobic polypeptides such as WT, PP and the like are connecting peptides, and the yield of the PKEK monomer after enzyme digestion is only equivalent to that of the non-connecting peptide group. Therefore, the connecting peptide provided by the invention can greatly improve the enzyme digestion yield of the PKEK monomer.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Zhuhai Federal pharmaceutical Co Ltd
<120> PKEK fusion protein, preparation method and application thereof
<160> 64
<170> SIPOSequenceListing 1.0
<210> 1
<211> 228
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> (AAK-PKEK)10
<400> 1
catatggctg ctaaaccgaa agaaaaagct gctaaaccga aagaaaaagc tgctaaaccg 60
aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac cgaaagaaaa agctgctaaa 120
ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga aaaagctgct 180
aaaccgaaag aaaaagctgc taaaccgaaa gaaaaataat gactcgag 228
<210> 2
<211> 127
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> KSI chaperones
<400> 2
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu
115 120 125
<210> 3
<211> 165
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> PagP chaperones
<400> 3
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu
165
<210> 4
<211> 96
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> TAF12 chaperone
<400> 4
Met Ser Pro Glu Asn Asn Gln Val Leu Thr Lys Lys Lys Leu Gln Asp
1 5 10 15
Leu Val Arg Glu Val Asp Pro Asn Glu Gln Leu Asp Glu Asp Val Glu
20 25 30
Glu Met Leu Leu Gln Ile Ala Asp Asp Phe Ile Glu Ser Val Val Thr
35 40 45
Ala Ala Cys Gln Leu Ala Arg His Arg Lys Ser Ser Thr Leu Glu Val
50 55 60
Lys Asp Val Gln Leu His Leu Glu Arg Gln Trp Asn Met Trp Ile Pro
65 70 75 80
Gly Phe Gly Ser Glu Glu Ile Arg Pro Tyr Lys Lys Ala Gln Met Leu
85 90 95
<210> 5
<211> 166
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> PaP3.30 chaperones
<400> 5
Met Leu Ser Lys Ser Leu Glu Asp His Glu Ala Gly Val Cys Pro Leu
1 5 10 15
Gly Cys Pro Tyr Cys Leu Ala Asp Tyr Glu Arg Ile Trp Gly Val Arg
20 25 30
Val Val Ser Ser Val Ala Ala Ser Asn Asp Lys Val Glu Val Asp Pro
35 40 45
Asn Gly Ile Lys Ala Gly Asp Pro Gly Ala Lys Leu Asp Lys Gly Lys
50 55 60
Val Asp Val Gly Ile Ile Phe Glu Ala Phe Pro Arg Ala Leu Tyr Ala
65 70 75 80
Val Ala Gln Val Ala Asn Phe Gly Ala Ser Lys Tyr Ser Arg Gly Gly
85 90 95
Trp Arg Phe Val Glu Asn Gly Ile Gln Arg Tyr Asp Ala Ala Phe Gly
100 105 110
Arg His Leu Leu Glu Arg His Lys Gly Glu Val Leu Asp Pro Gln Ser
115 120 125
Ser Leu Pro His Arg Tyr His Glu Val Trp Asn Ala Leu Ala Ala Leu
130 135 140
Glu Leu Val Ile Gln Gln Glu Glu Gly Ser Asn Gly Thr Ser Thr Gly
145 150 155 160
Ser Glu Gly Gln Met Leu
165
<210> 6
<211> 70
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> (AAK-PKEK)10
<400> 6
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
1 5 10 15
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
20 25 30
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
35 40 45
Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala
50 55 60
Ala Lys Pro Lys Glu Lys
65 70
<210> 7
<211> 41
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> K-(PKEK)10
<400> 7
Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu
1 5 10 15
Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu
20 25 30
Lys Pro Lys Glu Lys Pro Lys Glu Lys
35 40
<210> 8
<211> 81
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> K-(PKEK)20
<400> 8
Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu
1 5 10 15
Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu
20 25 30
Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu
35 40 45
Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu
50 55 60
Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu
65 70 75 80
Lys
<210> 9
<211> 132
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-K-PKEK
<400> 9
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Lys
115 120 125
Pro Lys Glu Lys
130
<210> 10
<211> 168
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-K-(PKEK)10
<400> 10
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Lys
115 120 125
Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys
130 135 140
Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys
145 150 155 160
Pro Lys Glu Lys Pro Lys Glu Lys
165
<210> 11
<211> 133
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-(AK-PKEK)
<400> 11
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Ala
115 120 125
Lys Pro Lys Glu Lys
130
<210> 12
<211> 187
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-(AK-PKEK)10
<400> 12
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Ala
115 120 125
Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu
130 135 140
Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro
145 150 155 160
Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala
165 170 175
Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys
180 185
<210> 13
<211> 187
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-(GK-PKEK)10
<400> 13
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Gly
115 120 125
Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu
130 135 140
Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro
145 150 155 160
Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly
165 170 175
Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys
180 185
<210> 14
<211> 197
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-(AAK-PKEK)10
<400> 14
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Ala
115 120 125
Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
130 135 140
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
145 150 155 160
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
165 170 175
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
180 185 190
Lys Pro Lys Glu Lys
195
<210> 15
<211> 197
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-(PPK-PKEK)10
<400> 15
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Pro
115 120 125
Pro Lys Pro Lys Glu Lys Pro Pro Lys Pro Lys Glu Lys Pro Pro Lys
130 135 140
Pro Lys Glu Lys Pro Pro Lys Pro Lys Glu Lys Pro Pro Lys Pro Lys
145 150 155 160
Glu Lys Pro Pro Lys Pro Lys Glu Lys Pro Pro Lys Pro Lys Glu Lys
165 170 175
Pro Pro Lys Pro Lys Glu Lys Pro Pro Lys Pro Lys Glu Lys Pro Pro
180 185 190
Lys Pro Lys Glu Lys
195
<210> 16
<211> 223
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-(AAGK-PKEK)12
<400> 16
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Ala
115 120 125
Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala
130 135 140
Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala
145 150 155 160
Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala
165 170 175
Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala
180 185 190
Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala
195 200 205
Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys
210 215 220
<210> 17
<211> 217
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-(AAGGK-PKEK)10
<400> 17
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Ala
115 120 125
Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu
130 135 140
Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro
145 150 155 160
Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly
165 170 175
Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala
180 185 190
Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys
195 200 205
Ala Ala Gly Gly Lys Pro Lys Glu Lys
210 215
<210> 18
<211> 262
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-(GGAAK-PKEK)15
<400> 18
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Gly
115 120 125
Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu
130 135 140
Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro
145 150 155 160
Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala
165 170 175
Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly
180 185 190
Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys
195 200 205
Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys
210 215 220
Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys
225 230 235 240
Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala
245 250 255
Ala Lys Pro Lys Glu Lys
260
<210> 19
<211> 227
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-( GGSASK-PKEK)10
<400> 19
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Gly
115 120 125
Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro
130 135 140
Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser
145 150 155 160
Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu
165 170 175
Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser
180 185 190
Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly
195 200 205
Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro
210 215 220
Lys Glu Lys
225
<210> 20
<211> 257
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-(AAGGSGAGK-PKEK)10
<400> 20
Met His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala
1 5 10 15
Ala Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp
20 25 30
Asp Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr
35 40 45
Ala Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala
50 55 60
Val Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe
65 70 75 80
Ala Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala
85 90 95
Pro Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile
100 105 110
Arg Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys Gln Met Leu Ala
115 120 125
Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly
130 135 140
Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala
145 150 155 160
Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro
165 170 175
Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys
180 185 190
Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
195 200 205
Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly
210 215 220
Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys
225 230 235 240
Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu
245 250 255
Lys
<210> 21
<211> 285
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> PagP-(AK-PKEK)20
<400> 21
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu
165 170 175
Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro
180 185 190
Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala
195 200 205
Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu
210 215 220
Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro
225 230 235 240
Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala
245 250 255
Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu
260 265 270
Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys
275 280 285
<210> 22
<211> 285
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> PagP-(GK-PKEK)20
<400> 22
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu
165 170 175
Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro
180 185 190
Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly
195 200 205
Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu
210 215 220
Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro
225 230 235 240
Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly
245 250 255
Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu
260 265 270
Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys
275 280 285
<210> 23
<211> 305
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> PagP-(AAK-PKEK)20
<400> 23
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
165 170 175
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
180 185 190
Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala
195 200 205
Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
210 215 220
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
225 230 235 240
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
245 250 255
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
260 265 270
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
275 280 285
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
290 295 300
Lys
305
<210> 24
<211> 305
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> PagP-(WTK-PKEK)20
<400> 24
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro
165 170 175
Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu
180 185 190
Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp
195 200 205
Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys
210 215 220
Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys
225 230 235 240
Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys
245 250 255
Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr
260 265 270
Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro
275 280 285
Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu
290 295 300
Lys
305
<210> 25
<211> 285
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> PagP-(AAGK-PKEK)15
<400> 25
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
165 170 175
Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
180 185 190
Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
195 200 205
Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
210 215 220
Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
225 230 235 240
Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
245 250 255
Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
260 265 270
Lys Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys
275 280 285
<210> 26
<211> 345
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> PagP-(AAGGK-PKEK)20
<400> 26
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala
165 170 175
Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys
180 185 190
Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys
195 200 205
Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys
210 215 220
Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly
225 230 235 240
Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala
245 250 255
Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu
260 265 270
Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro
275 280 285
Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly
290 295 300
Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys Ala Ala
305 310 315 320
Gly Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Lys Pro Lys Glu Lys
325 330 335
Ala Ala Gly Gly Lys Pro Lys Glu Lys
340 345
<210> 27
<211> 300
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> PagP-(GGAAK-PKEK)15
<400> 27
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly
165 170 175
Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys
180 185 190
Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys
195 200 205
Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys
210 215 220
Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala
225 230 235 240
Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly
245 250 255
Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu
260 265 270
Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro
275 280 285
Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys
290 295 300
<210> 28
<211> 365
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> PagP-(GGSASK-PKEK)20
<400> 28
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly
165 170 175
Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro
180 185 190
Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser
195 200 205
Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu
210 215 220
Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser
225 230 235 240
Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly
245 250 255
Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro
260 265 270
Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser
275 280 285
Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu
290 295 300
Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser
305 310 315 320
Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly
325 330 335
Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro
340 345 350
Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys
355 360 365
<210> 29
<211> 425
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> PagP-(AAGGSGAGK-PKEK)20
<400> 29
Met Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln
1 5 10 15
Thr Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr
20 25 30
Trp His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn
35 40 45
Glu Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys
50 55 60
Gly Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn
65 70 75 80
Lys Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro
85 90 95
Leu Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val
100 105 110
Thr Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro
115 120 125
Leu Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile
130 135 140
Pro Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe
145 150 155 160
Gln Phe Gln Met Leu Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys
165 170 175
Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala
180 185 190
Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly
195 200 205
Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala
210 215 220
Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro
225 230 235 240
Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys
245 250 255
Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly
260 265 270
Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly
275 280 285
Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys
290 295 300
Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu
305 310 315 320
Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala
325 330 335
Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser
340 345 350
Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly
355 360 365
Lys Pro Lys Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys
370 375 380
Glu Lys Ala Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala
385 390 395 400
Ala Gly Gly Ser Gly Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Gly
405 410 415
Ser Gly Ala Gly Lys Pro Lys Glu Lys
420 425
<210> 30
<211> 186
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> TAF12-(GGAAK-PKEK)10
<400> 30
Met Ser Pro Glu Asn Asn Gln Val Leu Thr Lys Lys Lys Leu Gln Asp
1 5 10 15
Leu Val Arg Glu Val Asp Pro Asn Glu Gln Leu Asp Glu Asp Val Glu
20 25 30
Glu Met Leu Leu Gln Ile Ala Asp Asp Phe Ile Glu Ser Val Val Thr
35 40 45
Ala Ala Cys Gln Leu Ala Arg His Arg Lys Ser Ser Thr Leu Glu Val
50 55 60
Lys Asp Val Gln Leu His Leu Glu Arg Gln Trp Asn Met Trp Ile Pro
65 70 75 80
Gly Phe Gly Ser Glu Glu Ile Arg Pro Tyr Lys Lys Ala Gln Met Leu
85 90 95
Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys
100 105 110
Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys
115 120 125
Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala
130 135 140
Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly
145 150 155 160
Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu
165 170 175
Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys
180 185
<210> 31
<211> 236
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> TAF12-(AAK-PKEK)20
<400> 31
Met Ser Pro Glu Asn Asn Gln Val Leu Thr Lys Lys Lys Leu Gln Asp
1 5 10 15
Leu Val Arg Glu Val Asp Pro Asn Glu Gln Leu Asp Glu Asp Val Glu
20 25 30
Glu Met Leu Leu Gln Ile Ala Asp Asp Phe Ile Glu Ser Val Val Thr
35 40 45
Ala Ala Cys Gln Leu Ala Arg His Arg Lys Ser Ser Thr Leu Glu Val
50 55 60
Lys Asp Val Gln Leu His Leu Glu Arg Gln Trp Asn Met Trp Ile Pro
65 70 75 80
Gly Phe Gly Ser Glu Glu Ile Arg Pro Tyr Lys Lys Ala Gln Met Leu
85 90 95
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
100 105 110
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
115 120 125
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
130 135 140
Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala
145 150 155 160
Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
165 170 175
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
180 185 190
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
195 200 205
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
210 215 220
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
225 230 235
<210> 32
<211> 256
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> PaP3.30-(GGAAK-PKEK)10
<400> 32
Met Leu Ser Lys Ser Leu Glu Asp His Glu Ala Gly Val Cys Pro Leu
1 5 10 15
Gly Cys Pro Tyr Cys Leu Ala Asp Tyr Glu Arg Ile Trp Gly Val Arg
20 25 30
Val Val Ser Ser Val Ala Ala Ser Asn Asp Lys Val Glu Val Asp Pro
35 40 45
Asn Gly Ile Lys Ala Gly Asp Pro Gly Ala Lys Leu Asp Lys Gly Lys
50 55 60
Val Asp Val Gly Ile Ile Phe Glu Ala Phe Pro Arg Ala Leu Tyr Ala
65 70 75 80
Val Ala Gln Val Ala Asn Phe Gly Ala Ser Lys Tyr Ser Arg Gly Gly
85 90 95
Trp Arg Phe Val Glu Asn Gly Ile Gln Arg Tyr Asp Ala Ala Phe Gly
100 105 110
Arg His Leu Leu Glu Arg His Lys Gly Glu Val Leu Asp Pro Gln Ser
115 120 125
Ser Leu Pro His Arg Tyr His Glu Val Trp Asn Ala Leu Ala Ala Leu
130 135 140
Glu Leu Val Ile Gln Gln Glu Glu Gly Ser Asn Gly Thr Ser Thr Gly
145 150 155 160
Ser Glu Gly Gln Met Leu Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly
165 170 175
Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu
180 185 190
Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro
195 200 205
Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala
210 215 220
Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly
225 230 235 240
Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys
245 250 255
<210> 33
<211> 306
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> PaP3.30-(AAK-PKEK)20
<400> 33
Met Leu Ser Lys Ser Leu Glu Asp His Glu Ala Gly Val Cys Pro Leu
1 5 10 15
Gly Cys Pro Tyr Cys Leu Ala Asp Tyr Glu Arg Ile Trp Gly Val Arg
20 25 30
Val Val Ser Ser Val Ala Ala Ser Asn Asp Lys Val Glu Val Asp Pro
35 40 45
Asn Gly Ile Lys Ala Gly Asp Pro Gly Ala Lys Leu Asp Lys Gly Lys
50 55 60
Val Asp Val Gly Ile Ile Phe Glu Ala Phe Pro Arg Ala Leu Tyr Ala
65 70 75 80
Val Ala Gln Val Ala Asn Phe Gly Ala Ser Lys Tyr Ser Arg Gly Gly
85 90 95
Trp Arg Phe Val Glu Asn Gly Ile Gln Arg Tyr Asp Ala Ala Phe Gly
100 105 110
Arg His Leu Leu Glu Arg His Lys Gly Glu Val Leu Asp Pro Gln Ser
115 120 125
Ser Leu Pro His Arg Tyr His Glu Val Trp Asn Ala Leu Ala Ala Leu
130 135 140
Glu Leu Val Ile Gln Gln Glu Glu Gly Ser Asn Gly Thr Ser Thr Gly
145 150 155 160
Ser Glu Gly Gln Met Leu Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
165 170 175
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
180 185 190
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
195 200 205
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
210 215 220
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
225 230 235 240
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
245 250 255
Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala
260 265 270
Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
275 280 285
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
290 295 300
Glu Lys
305
<210> 34
<211> 376
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<223> PaP3.30-(AAK-PKEK)30
<400> 34
Met Leu Ser Lys Ser Leu Glu Asp His Glu Ala Gly Val Cys Pro Leu
1 5 10 15
Gly Cys Pro Tyr Cys Leu Ala Asp Tyr Glu Arg Ile Trp Gly Val Arg
20 25 30
Val Val Ser Ser Val Ala Ala Ser Asn Asp Lys Val Glu Val Asp Pro
35 40 45
Asn Gly Ile Lys Ala Gly Asp Pro Gly Ala Lys Leu Asp Lys Gly Lys
50 55 60
Val Asp Val Gly Ile Ile Phe Glu Ala Phe Pro Arg Ala Leu Tyr Ala
65 70 75 80
Val Ala Gln Val Ala Asn Phe Gly Ala Ser Lys Tyr Ser Arg Gly Gly
85 90 95
Trp Arg Phe Val Glu Asn Gly Ile Gln Arg Tyr Asp Ala Ala Phe Gly
100 105 110
Arg His Leu Leu Glu Arg His Lys Gly Glu Val Leu Asp Pro Gln Ser
115 120 125
Ser Leu Pro His Arg Tyr His Glu Val Trp Asn Ala Leu Ala Ala Leu
130 135 140
Glu Leu Val Ile Gln Gln Glu Glu Gly Ser Asn Gly Thr Ser Thr Gly
145 150 155 160
Ser Glu Gly Gln Met Leu Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
165 170 175
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
180 185 190
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
195 200 205
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
210 215 220
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
225 230 235 240
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
245 250 255
Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala
260 265 270
Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
275 280 285
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
290 295 300
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
305 310 315 320
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
325 330 335
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
340 345 350
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
355 360 365
Lys Ala Ala Lys Pro Lys Glu Lys
370 375
<210> 35
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> T7 promoter primer
<400> 35
taatacgact cactataggg 20
<210> 36
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> T7 terminator primer
<400> 36
tgctagttat tgctcagcgg 20
<210> 37
<211> 870
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> PagP-(AK-PKEK)20
<400> 37
catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag 60
cagccggaac actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac 120
gacaaagaaa aaaccgaccg ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct 180
cgttgggacg aaaaaggtaa ctggcacggt ctgtacgcta tggctttcaa agactcttgg 240
aacaaatggg aaccgatcgc tggttacggt tgggaatcta cctggcgtcc gctggctgac 300
gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga caactggaac 360
tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc 420
cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt 480
ttccagttcc agatgctggc taaaccgaaa gaaaaagcta aaccgaaaga aaaagctaaa 540
ccgaaagaaa aagctaaacc gaaagaaaaa gctaaaccga aagaaaaagc taaaccgaaa 600
gaaaaagcta aaccgaaaga aaaagctaaa ccgaaagaaa aagctaaacc gaaagaaaaa 660
gctaaaccga aagaaaaagc taaaccgaaa gaaaaagcta aaccgaaaga aaaagctaaa 720
ccgaaagaaa aagctaaacc gaaagaaaaa gctaaaccga aagaaaaagc taaaccgaaa 780
gaaaaagcta aaccgaaaga aaaagctaaa ccgaaagaaa aagctaaacc gaaagaaaaa 840
gctaaaccga aagaaaaata atgactcgag 870
<210> 38
<211> 573
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> TAF12-(GGAAK-PKEK)10
<400> 38
catatgtctc cggaaaacaa ccaggttctg accaaaaaaa aactgcagga cctggttcgt 60
gaagttgacc cgaacgaaca gctggacgaa gacgttgaag aaatgctgct gcagatcgct 120
gacgacttca tcgaatctgt tgttaccgct gcttgccagc tggctcgtca ccgtaaatct 180
tctaccctgg aagttaaaga cgttcagctg cacctggaac gtcagtggaa catgtggatc 240
ccgggtttcg gttctgaaga aatccgtccg tacaaaaaag ctcagatgct gggtggtgct 300
gctaaaccga aagaaaaagg tggtgctgct aaaccgaaag aaaaaggtgg tgctgctaaa 360
ccgaaagaaa aaggtggtgc tgctaaaccg aaagaaaaag gtggtgctgc taaaccgaaa 420
gaaaaaggtg gtgctgctaa accgaaagaa aaaggtggtg ctgctaaacc gaaagaaaaa 480
ggtggtgctg ctaaaccgaa agaaaaaggt ggtgctgcta aaccgaaaga aaaaggtggt 540
gctgctaaac cgaaagaaaa ataatgactc gag 573
<210> 39
<211> 783
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> PaP3.30-(GGAAK-PKEK)10
<400> 39
catatgctgt ctaaatctct ggaagaccac gaagctggtg tttgcccgct gggttgcccg 60
tactgcctgg ctgactacga acgtatctgg ggtgttcgtg ttgtttcttc tgttgctgct 120
tctaacgaca aagttgaagt tgacccgaac ggtatcaaag ctggtgaccc gggtgctaaa 180
ctggacaaag gtaaagttga cgttggtatc atcttcgaag ctttcccgcg tgctctgtac 240
gctgttgctc aggttgctaa cttcggtgct tctaaatact ctcgtggtgg ttggcgtttc 300
gttgaaaacg gtatccagcg ttacgacgct gctttcggtc gtcacctgct ggaacgtcac 360
aaaggtgaag ttctggaccc gcagtcttct ctgccgcacc gttaccacga agtttggaac 420
gctctggctg ctctggaact ggttatccag caggaagaag gttctaacgg tacctctacc 480
ggttctgaag gtcagatgct gggtggtgct gctaaaccga aagaaaaagg tggtgctgct 540
aaaccgaaag aaaaaggtgg tgctgctaaa ccgaaagaaa aaggtggtgc tgctaaaccg 600
aaagaaaaag gtggtgctgc taaaccgaaa gaaaaaggtg gtgctgctaa accgaaagaa 660
aaaggtggtg ctgctaaacc gaaagaaaaa ggtggtgctg ctaaaccgaa agaaaaaggt 720
ggtgctgcta aaccgaaaga aaaaggtggt gctgctaaac cgaaagaaaa ataatgactc 780
gag 783
<210> 40
<211> 606
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-(AAK-PKEK)10
<400> 40
catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat 60
gccggcgatc tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc 120
gtgggttccg agcccaggtc cggtacggct gcgattcgtg agttttacgc caactcgctc 180
aaactgcctt tggcggtgga gctgacgcag gaggtacgcg cggtcgccaa cgaagcggcc 240
ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga ccgtagttgc gcccatcgat 300
cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt tggcgagaag 360
aatattcacg catgccagat gctggctgct aaaccgaaag aaaaagctgc taaaccgaaa 420
gaaaaagctg ctaaaccgaa agaaaaagct gctaaaccga aagaaaaagc tgctaaaccg 480
aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac cgaaagaaaa agctgctaaa 540
ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga aaaataatga 600
ctcgag 606
<210> 41
<211> 684
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-(AAGK-PKEK)12
<400> 41
catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat 60
gccggcgatc tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc 120
gtgggttccg agcccaggtc cggtacggct gcgattcgtg agttttacgc caactcgctc 180
aaactgcctt tggcggtgga gctgacgcag gaggtacgcg cggtcgccaa cgaagcggcc 240
ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga ccgtagttgc gcccatcgat 300
cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt tggcgagaag 360
aatattcacg catgccagat gctggctgct ggtaaaccga aagaaaaagc tgctggtaaa 420
ccgaaagaaa aagctgctgg taaaccgaaa gaaaaagctg ctggtaaacc gaaagaaaaa 480
gctgctggta aaccgaaaga aaaagctgct ggtaaaccga aagaaaaagc tgctggtaaa 540
ccgaaagaaa aagctgctgg taaaccgaaa gaaaaagctg ctggtaaacc gaaagaaaaa 600
gctgctggta aaccgaaaga aaaagctgct ggtaaaccga aagaaaaagc tgctggtaaa 660
ccgaaagaaa aataatgact cgag 684
<210> 42
<211> 801
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-(GGAAK-PKEK)15
<400> 42
catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat 60
gccggcgatc tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc 120
gtgggttccg agcccaggtc cggtacggct gcgattcgtg agttttacgc caactcgctc 180
aaactgcctt tggcggtgga gctgacgcag gaggtacgcg cggtcgccaa cgaagcggcc 240
ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga ccgtagttgc gcccatcgat 300
cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt tggcgagaag 360
aatattcacg catgccagat gctgggtggt gctgctaaac cgaaagaaaa aggtggtgct 420
gctaaaccga aagaaaaagg tggtgctgct aaaccgaaag aaaaaggtgg tgctgctaaa 480
ccgaaagaaa aaggtggtgc tgctaaaccg aaagaaaaag gtggtgctgc taaaccgaaa 540
gaaaaaggtg gtgctgctaa accgaaagaa aaaggtggtg ctgctaaacc gaaagaaaaa 600
ggtggtgctg ctaaaccgaa agaaaaaggt ggtgctgcta aaccgaaaga aaaaggtggt 660
gctgctaaac cgaaagaaaa aggtggtgct gctaaaccga aagaaaaagg tggtgctgct 720
aaaccgaaag aaaaaggtgg tgctgctaaa ccgaaagaaa aaggtggtgc tgctaaaccg 780
aaagaaaaat aatgactcga g 801
<210> 43
<211> 930
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> PagP-(AAK-PKEK)20
<400> 43
catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag 60
cagccggaac actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac 120
gacaaagaaa aaaccgaccg ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct 180
cgttgggacg aaaaaggtaa ctggcacggt ctgtacgcta tggctttcaa agactcttgg 240
aacaaatggg aaccgatcgc tggttacggt tgggaatcta cctggcgtcc gctggctgac 300
gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga caactggaac 360
tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc 420
cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt 480
ttccagttcc agatgctggc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa 540
gctgctaaac cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa 600
aaagctgcta aaccgaaaga aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa 660
gaaaaagctg ctaaaccgaa agaaaaagct gctaaaccga aagaaaaagc tgctaaaccg 720
aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac cgaaagaaaa agctgctaaa 780
ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga aaaagctgct 840
aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct 900
gctaaaccga aagaaaaata atgactcgag 930
<210> 44
<211> 870
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> PagP-(AAGK-PKEK)15
<400> 44
catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag 60
cagccggaac actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac 120
gacaaagaaa aaaccgaccg ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct 180
cgttgggacg aaaaaggtaa ctggcacggt ctgtacgcta tggctttcaa agactcttgg 240
aacaaatggg aaccgatcgc tggttacggt tgggaatcta cctggcgtcc gctggctgac 300
gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga caactggaac 360
tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc 420
cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt 480
ttccagttcc agatgctggc tgctggtaaa ccgaaagaaa aagctgctgg taaaccgaaa 540
gaaaaagctg ctggtaaacc gaaagaaaaa gctgctggta aaccgaaaga aaaagctgct 600
ggtaaaccga aagaaaaagc tgctggtaaa ccgaaagaaa aagctgctgg taaaccgaaa 660
gaaaaagctg ctggtaaacc gaaagaaaaa gctgctggta aaccgaaaga aaaagctgct 720
ggtaaaccga aagaaaaagc tgctggtaaa ccgaaagaaa aagctgctgg taaaccgaaa 780
gaaaaagctg ctggtaaacc gaaagaaaaa gctgctggta aaccgaaaga aaaagctgct 840
ggtaaaccga aagaaaaata atgactcgag 870
<210> 45
<211> 1050
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> PagP-(AAGGK-PKEK)20
<400> 45
catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag 60
cagccggaac actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac 120
gacaaagaaa aaaccgaccg ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct 180
cgttgggacg aaaaaggtaa ctggcacggt ctgtacgcta tggctttcaa agactcttgg 240
aacaaatggg aaccgatcgc tggttacggt tgggaatcta cctggcgtcc gctggctgac 300
gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga caactggaac 360
tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc 420
cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt 480
ttccagttcc agatgctggc tgctggtggt aaaccgaaag aaaaagctgc tggtggtaaa 540
ccgaaagaaa aagctgctgg tggtaaaccg aaagaaaaag ctgctggtgg taaaccgaaa 600
gaaaaagctg ctggtggtaa accgaaagaa aaagctgctg gtggtaaacc gaaagaaaaa 660
gctgctggtg gtaaaccgaa agaaaaagct gctggtggta aaccgaaaga aaaagctgct 720
ggtggtaaac cgaaagaaaa agctgctggt ggtaaaccga aagaaaaagc tgctggtggt 780
aaaccgaaag aaaaagctgc tggtggtaaa ccgaaagaaa aagctgctgg tggtaaaccg 840
aaagaaaaag ctgctggtgg taaaccgaaa gaaaaagctg ctggtggtaa accgaaagaa 900
aaagctgctg gtggtaaacc gaaagaaaaa gctgctggtg gtaaaccgaa agaaaaagct 960
gctggtggta aaccgaaaga aaaagctgct ggtggtaaac cgaaagaaaa agctgctggt 1020
ggtaaaccga aagaaaaata atgactcgag 1050
<210> 46
<211> 915
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> PagP-(GGAAK-PKEK)15
<400> 46
catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag 60
cagccggaac actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac 120
gacaaagaaa aaaccgaccg ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct 180
cgttgggacg aaaaaggtaa ctggcacggt ctgtacgcta tggctttcaa agactcttgg 240
aacaaatggg aaccgatcgc tggttacggt tgggaatcta cctggcgtcc gctggctgac 300
gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga caactggaac 360
tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc 420
cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt 480
ttccagttcc agatgctggg tggtgctgct aaaccgaaag aaaaaggtgg tgctgctaaa 540
ccgaaagaaa aaggtggtgc tgctaaaccg aaagaaaaag gtggtgctgc taaaccgaaa 600
gaaaaaggtg gtgctgctaa accgaaagaa aaaggtggtg ctgctaaacc gaaagaaaaa 660
ggtggtgctg ctaaaccgaa agaaaaaggt ggtgctgcta aaccgaaaga aaaaggtggt 720
gctgctaaac cgaaagaaaa aggtggtgct gctaaaccga aagaaaaagg tggtgctgct 780
aaaccgaaag aaaaaggtgg tgctgctaaa ccgaaagaaa aaggtggtgc tgctaaaccg 840
aaagaaaaag gtggtgctgc taaaccgaaa gaaaaaggtg gtgctgctaa accgaaagaa 900
aaataatgac tcgag 915
<210> 47
<211> 141
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> K-(PKEK)10
<400> 47
1 catatgaaac cgaaagaaaa accgaaagaa aaaccgaaag aaaaaccgaa agaaaaaccg aaagaaaaac
71 cgaaagaaaa accgaaagaa aaaccgaaag aaaaaccgaa agaaaaaccg aaagaaaaat aatgactcga
141 g
<210> 48
<211> 261
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> K-(PKEK)20
<400> 48
1 catatgaaac cgaaagaaaa accgaaagaa aaaccgaaag aaaaaccgaa agaaaaaccg aaagaaaaac
71 cgaaagaaaa accgaaagaa aaaccgaaag aaaaaccgaa agaaaaaccg aaagaaaaac cgaaagaaaa
141 accgaaagaa aaaccgaaag aaaaaccgaa agaaaaaccg aaagaaaaac cgaaagaaaa accgaaagaa
211 aaaccgaaag aaaaaccgaa agaaaaaccg aaagaaaaat aatgactcga g
<210> 49
<211> 411
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-K-PKEK
<400> 49
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctgaaaccg aaagaaaaat aatgactcga g
<210> 50
<211> 519
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-K-（PKEK）10
<400> 49
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctgaaaccg aaagaaaaac cgaaagaaaa accgaaagaa
421 aaaccgaaag aaaaaccgaa agaaaaaccg aaagaaaaac cgaaagaaaa accgaaagaa aaaccgaaag
491 aaaaaccgaa agaaaaataa tgactcgag
<210> 51
<211> 414
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-AK-PKEK
<400> 51
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctggctaaa ccgaaagaaa aataatgact cgag
<210> 52
<211> 576
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-（AK-PKEK）10
<400> 52
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctggctaaa ccgaaagaaa aagctaaacc gaaagaaaaa
421 gctaaaccga aagaaaaagc taaaccgaaa gaaaaagcta aaccgaaaga aaaagctaaa ccgaaagaaa
491 aagctaaacc gaaagaaaaa gctaaaccga aagaaaaagc taaaccgaaa gaaaaagcta aaccgaaaga
561 aaaataatga ctcgag
<210> 53
<211> 576
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-（GK-PKEK）10
<400> 53
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctgggtaaa ccgaaagaaa aaggtaaacc gaaagaaaaa
421 ggtaaaccga aagaaaaagg taaaccgaaa gaaaaaggta aaccgaaaga aaaaggtaaa ccgaaagaaa
491 aaggtaaacc gaaagaaaaa ggtaaaccga aagaaaaagg taaaccgaaa gaaaaaggta aaccgaaaga
561 aaaataatga ctcgag
<210> 54
<211> 606
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-（PPK-PKEK）10
<400> 54
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctgccgccg aaaccgaaag aaaaaccgcc gaaaccgaaa
421 gaaaaaccgc cgaaaccgaa agaaaaaccg ccgaaaccga aagaaaaacc gccgaaaccg aaagaaaaac
491 cgccgaaacc gaaagaaaaa ccgccgaaac cgaaagaaaa accgccgaaa ccgaaagaaa aaccgccgaa
561 accgaaagaa aaaccgccga aaccgaaaga aaaataatga ctcgag
<210> 55
<211> 666
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-（AAGGK-PKEK）10
<400> 55
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctggctgct ggtggtaaac cgaaagaaaa agctgctggt
421 ggtaaaccga aagaaaaagc tgctggtggt aaaccgaaag aaaaagctgc tggtggtaaa ccgaaagaaa
491 aagctgctgg tggtaaaccg aaagaaaaag ctgctggtgg taaaccgaaa gaaaaagctg ctggtggtaa
561 accgaaagaa aaagctgctg gtggtaaacc gaaagaaaaa gctgctggtg gtaaaccgaa agaaaaagct
631 gctggtggta aaccgaaaga aaaataatga ctcgag
<210> 56
<211> 696
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-（GGSASK-PKEK）10
<400> 56
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctgggtggt tctgcttcta aaccgaaaga aaaaggtggt
421 tctgcttcta aaccgaaaga aaaaggtggt tctgcttcta aaccgaaaga aaaaggtggt tctgcttcta
491 aaccgaaaga aaaaggtggt tctgcttcta aaccgaaaga aaaaggtggt tctgcttcta aaccgaaaga
561 aaaaggtggt tctgcttcta aaccgaaaga aaaaggtggt tctgcttcta aaccgaaaga aaaaggtggt
631 tctgcttcta aaccgaaaga aaaaggtggt tctgcttcta aaccgaaaga aaaataatga ctcgag
<210> 57
<211> 915
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> KSI-（AAGGSGAGK-PKEK）10
<400> 57
1 catatgcata ccccagaaca catcaccgcc gtggtacagc gctttgtggc tgcgctcaat gccggcgatc
71 tggacggcat cgtcgcgctg tttgccgatg acgccacggt ggaagacccc gtgggttccg agcccaggtc
141 cggtacggct gcgattcgtg agttttacgc caactcgctc aaactgcctt tggcggtgga gctgacgcag
211 gaggtacgcg cggtcgccaa cgaagcggcc ttcgctttca ccgtcagctt cgagtatcag ggccgcaaga
281 ccgtagttgc gcccatcgat cactttcgct tcaatggcgc cggcaaggtg gtgagcatcc gcgccttgtt
351 tggcgagaag aatattcacg catgccagat gctggctgct ggtggttctg gtgctggtaa accgaaagaa
421 aaagctgctg gtggttctgg tgctggtaaa ccgaaagaaa aagctgctgg tggttctggt gctggtaaac
491 cgaaagaaaa agctgctggt ggttctggtg ctggtaaacc gaaagaaaaa gctgctggtg gttctggtgc
561 tggtaaaccg aaagaaaaag ctgctggtgg ttctggtgct ggtaaaccga aagaaaaagc tgctggtggt
631 tctggtgctg gtaaaccgaa agaaaaagct gctggtggtt ctggtgctgg taaaccgaaa gaaaaagctg
701 ctggtggttc tggtgctggt aaaccgaaag aaaaagctgc tggtggttct ggtgctggta aaccgaaaga
771 aaaataatga ctcgag
<210> 58
<211> 870
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> PagP-（GK-PKEK）20
<400> 58
1 catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag cagccggaac
71 actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac gacaaagaaa aaaccgaccg
141 ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct cgttgggacg aaaaaggtaa ctggcacggt
211 ctgtacgcta tggctttcaa agactcttgg aacaaatggg aaccgatcgc tggttacggt tgggaatcta
281 cctggcgtcc gctggctgac gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga
351 caactggaac tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc
421 cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt ttccagttcc
491 agatgctggg taaaccgaaa gaaaaaggta aaccgaaaga aaaaggtaaa ccgaaagaaa aaggtaaacc
561 gaaagaaaaa ggtaaaccga aagaaaaagg taaaccgaaa gaaaaaggta aaccgaaaga aaaaggtaaa
631 ccgaaagaaa aaggtaaacc gaaagaaaaa ggtaaaccga aagaaaaagg taaaccgaaa gaaaaaggta
701 aaccgaaaga aaaaggtaaa ccgaaagaaa aaggtaaacc gaaagaaaaa ggtaaaccga aagaaaaagg
771 taaaccgaaa gaaaaaggta aaccgaaaga aaaaggtaaa ccgaaagaaa aaggtaaacc gaaagaaaaa
841 ggtaaaccga aagaaaaata atgactcgag
<210> 59
<211> 930
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> PagP-（WTK-PKEK）20
<400> 59
1 catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag cagccggaac
71 actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac gacaaagaaa aaaccgaccg
141 ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct cgttgggacg aaaaaggtaa ctggcacggt
211 ctgtacgcta tggctttcaa agactcttgg aacaaatggg aaccgatcgc tggttacggt tgggaatcta
281 cctggcgtcc gctggctgac gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga
351 caactggaac tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc
421 cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt ttccagttcc
491 agatgctgtg gaccaaaccg aaagaaaaat ggaccaaacc gaaagaaaaa tggaccaaac cgaaagaaaa
561 atggaccaaa ccgaaagaaa aatggaccaa accgaaagaa aaatggacca aaccgaaaga aaaatggacc
631 aaaccgaaag aaaaatggac caaaccgaaa gaaaaatgga ccaaaccgaa agaaaaatgg accaaaccga
701 aagaaaaatg gaccaaaccg aaagaaaaat ggaccaaacc gaaagaaaaa tggaccaaac cgaaagaaaa
771 atggaccaaa ccgaaagaaa aatggaccaa accgaaagaa aaatggacca aaccgaaaga aaaatggacc
841 aaaccgaaag aaaaatggac caaaccgaaa gaaaaatgga ccaaaccgaa agaaaaatgg accaaaccga
911 aagaaaaata atgactcgag
<210> 60
<211> 1110
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> PagP-（GGSASK-PKEK）20
<400> 60
1 catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag cagccggaac
71 actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac gacaaagaaa aaaccgaccg
141 ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct cgttgggacg aaaaaggtaa ctggcacggt
211 ctgtacgcta tggctttcaa agactcttgg aacaaatggg aaccgatcgc tggttacggt tgggaatcta
281 cctggcgtcc gctggctgac gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga
351 caactggaac tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc
421 cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt ttccagttcc
491 agatgctggg tggttctgct tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaagg
561 tggttctgct tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaagg tggttctgct
631 tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaagg tggttctgct tctaaaccga
701 aagaaaaagg tggttctgct tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaagg
771 tggttctgct tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaagg tggttctgct
841 tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaagg tggttctgct tctaaaccga
911 aagaaaaagg tggttctgct tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaagg
981 tggttctgct tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaagg tggttctgct
1051 tctaaaccga aagaaaaagg tggttctgct tctaaaccga aagaaaaata atgactcgag
<210> 61
<211> 1290
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> PagP-（AAGGSGAGK-PKEK）20
<400> 61
1 catatgaacg ctgacgaatg gatgaccacc ttccgtgaaa acatcgctca gacctggcag cagccggaac
71 actacgacct gtacatcccg gctatcacct ggcacgctcg tttcgcttac gacaaagaaa aaaccgaccg
141 ttacaacgaa cgtccgtggg gtggtggttt cggtctgtct cgttgggacg aaaaaggtaa ctggcacggt
211 ctgtacgcta tggctttcaa agactcttgg aacaaatggg aaccgatcgc tggttacggt tgggaatcta
281 cctggcgtcc gctggctgac gaaaacttcc acctgggtct gggtttcacc gctggtgtta ccgctcgtga
351 caactggaac tacatcccgc tgccggttct gctgccgctg gcttctgttg gttacggtcc ggttaccttc
421 cagatgacct acatcccggg tacctacaac aacggtaacg tttacttcgc ttggatgcgt ttccagttcc
491 agatgctggc tgctggtggt tctggtgctg gtaaaccgaa agaaaaagct gctggtggtt ctggtgctgg
561 taaaccgaaa gaaaaagctg ctggtggttc tggtgctggt aaaccgaaag aaaaagctgc tggtggttct
631 ggtgctggta aaccgaaaga aaaagctgct ggtggttctg gtgctggtaa accgaaagaa aaagctgctg
701 gtggttctgg tgctggtaaa ccgaaagaaa aagctgctgg tggttctggt gctggtaaac cgaaagaaaa
771 agctgctggt ggttctggtg ctggtaaacc gaaagaaaaa gctgctggtg gttctggtgc tggtaaaccg
841 aaagaaaaag ctgctggtgg ttctggtgct ggtaaaccga aagaaaaagc tgctggtggt tctggtgctg
911 gtaaaccgaa agaaaaagct gctggtggtt ctggtgctgg taaaccgaaa gaaaaagctg ctggtggttc
981 tggtgctggt aaaccgaaag aaaaagctgc tggtggttct ggtgctggta aaccgaaaga aaaagctgct
1051 ggtggttctg gtgctggtaa accgaaagaa aaagctgctg gtggttctgg tgctggtaaa ccgaaagaaa
1121 aagctgctgg tggttctggt gctggtaaac cgaaagaaaa agctgctggt ggttctggtg ctggtaaacc
1191 gaaagaaaaa gctgctggtg gttctggtgc tggtaaaccg aaagaaaaag ctgctggtgg ttctggtgct
1261 ggtaaaccga aagaaaaata atgactcgag
<210> 62
<211> 723
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> TAF12-（AAK-PKEK）20
<400> 62
1 catatgtctc cggaaaacaa ccaggttctg accaaaaaaa aactgcagga cctggttcgt gaagttgacc
71 cgaacgaaca gctggacgaa gacgttgaag aaatgctgct gcagatcgct gacgacttca tcgaatctgt
141 tgttaccgct gcttgccagc tggctcgtca ccgtaaatct tctaccctgg aagttaaaga cgttcagctg
211 cacctggaac gtcagtggaa catgtggatc ccgggtttcg gttctgaaga aatccgtccg tacaaaaaag
281 ctcagatgct ggctgctaaa ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga
351 aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct
421 gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac
491 cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga
561 aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct
631 gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac
701 cgaaagaaaa ataatgactc gag
<210> 63
<211> 933
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> PagP3.30-（AAK-PKEK）20
<400> 63
1 catatgctgt ctaaatctct ggaagaccac gaagctggtg tttgcccgct gggttgcccg tactgcctgg
71 ctgactacga acgtatctgg ggtgttcgtg ttgtttcttc tgttgctgct tctaacgaca aagttgaagt
141 tgacccgaac ggtatcaaag ctggtgaccc gggtgctaaa ctggacaaag gtaaagttga cgttggtatc
211 atcttcgaag ctttcccgcg tgctctgtac gctgttgctc aggttgctaa cttcggtgct tctaaatact
281 ctcgtggtgg ttggcgtttc gttgaaaacg gtatccagcg ttacgacgct gctttcggtc gtcacctgct
351 ggaacgtcac aaaggtgaag ttctggaccc gcagtcttct ctgccgcacc gttaccacga agtttggaac
421 gctctggctg ctctggaact ggttatccag caggaagaag gttctaacgg tacctctacc ggttctgaag
491 gtcagatgct ggctgctaaa ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga
561 aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct
631 gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac
701 cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga
771 aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct
841 gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac
911 cgaaagaaaa ataatgactc gag
<210> 64
<211> 1143
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<223> PagP3.30-（AAK-PKEK）30
<400> 64
1 catatgctgt ctaaatctct ggaagaccac gaagctggtg tttgcccgct gggttgcccg tactgcctgg
71 ctgactacga acgtatctgg ggtgttcgtg ttgtttcttc tgttgctgct tctaacgaca aagttgaagt
141 tgacccgaac ggtatcaaag ctggtgaccc gggtgctaaa ctggacaaag gtaaagttga cgttggtatc
211 atcttcgaag ctttcccgcg tgctctgtac gctgttgctc aggttgctaa cttcggtgct tctaaatact
281 ctcgtggtgg ttggcgtttc gttgaaaacg gtatccagcg ttacgacgct gctttcggtc gtcacctgct
351 ggaacgtcac aaaggtgaag ttctggaccc gcagtcttct ctgccgcacc gttaccacga agtttggaac
421 gctctggctg ctctggaact ggttatccag caggaagaag gttctaacgg tacctctacc ggttctgaag
491 gtcagatgct ggctgctaaa ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga
561 aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct
631 gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac
701 cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga
771 aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct
841 gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac
911 cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga
981 aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct
1051 gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac
1121 cgaaagaaaa ataatgactc gag

Claims (10)

1. A PKEK fusion protein, comprising: comprises a protein formed by connecting a plurality of flexible connecting peptides and whitening polypeptide PKEK in series in sequence, and the structural formula is as follows: (B-PKEK)n(ii) a Wherein: b is flexible connecting peptide, and n is an integer of 1-30;
the flexible connecting peptide is Xm---X2-X1The peptide sequence shown, m is an integer from 0 to 10; x1As protease cleavage site, X2To XmIs a flexible segment;
the amino acid in the flexible fragment cannot be Pro, Thr or Trp.
2. The PKEK fusion protein of claim 1, wherein: also included are chaperonins linked to a linker peptide of the formula: a- (B-PKEK)n(ii) a Wherein: a is chaperonin, B is flexible connecting peptide, and n is an integer of 1-30.
3. The PKEK fusion protein of claim 2, wherein: the chaperone protein is KSI, PagP, PaP3.30, TAF12, mKSI, mPAgP, mPAP3.30 or mTAF12, and m represents a mutant.
4. The PKEK fusion protein according to any of claims 1 to 3, characterized in that:
the protease restriction site is Arg or Lys;
the amino acid in the flexible segment is Gly, Ala or Ser.
5. The PKEK fusion protein of claim 4, wherein: the flexible connecting peptide is AK, GK, AAK, AAGK, AAGGK, GGAAK, GGSASK or AAGGSGAGK.
6. A gene encoding the PKEK fusion protein of any of claims 1 to 5.
7. A recombinant vector characterized by: comprising the gene of claim 6.
8. A recombinant expression strain comprising the recombinant vector according to claim 7.
9. A preparation method for producing whitening polypeptide PEKE by using a genetic engineering technology is characterized by comprising the following steps:
s1, obtaining a recombinant vector containing the gene of claim 6, wherein the encoded fusion protein is A- (B-PKEK)nWherein: a is chaperonin; b is flexible connecting peptide; n is an integer of 1 to 30;
s2, transferring the recombinant vector obtained in the step S1 into a genetic engineering strain to obtain a recombinant expression strain; fermenting and culturing the recombinant expression strain, and collecting inclusion bodies of the fusion protein;
s3, dissolving and enzyme cutting the inclusion body of the fusion protein, and separating and purifying to obtain the active whitening polypeptide PKEK.
10. The use of the method of claim 9 for producing PEKE as a whitening polypeptide in an industrial process.
CN201911075149.5A 2019-11-06 2019-11-06 PKEK fusion protein and preparation method and application thereof Active CN112759653B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201911075149.5A CN112759653B (en) 2019-11-06 2019-11-06 PKEK fusion protein and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201911075149.5A CN112759653B (en) 2019-11-06 2019-11-06 PKEK fusion protein and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN112759653A true CN112759653A (en) 2021-05-07
CN112759653B CN112759653B (en) 2023-06-23

Family

ID=75692792

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201911075149.5A Active CN112759653B (en) 2019-11-06 2019-11-06 PKEK fusion protein and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN112759653B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113616807A (en) * 2021-07-26 2021-11-09 中山大学 Mitochondrial-targeted polypeptide and preparation method and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080206160A1 (en) * 2007-01-05 2008-08-28 Helix Biomedix, Inc. Short bio-active peptides for cellular and immunological modulation
CN101878019A (en) * 2007-11-30 2010-11-03 赢创高施米特有限公司 Personal care and cosmetic composition containing tetrapeptides with the motifs gx1x2g, px1x2p, or px1x2k
CN107652355A (en) * 2017-10-26 2018-02-02 陕西慧康生物科技有限责任公司 The liquid-phase synthesis process of skin lightening peptide
CN108220315A (en) * 2016-12-22 2018-06-29 珠海冀百康生物科技有限公司 The preparation method and fusion protein of a kind of small molecular protein or polypeptide

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080206160A1 (en) * 2007-01-05 2008-08-28 Helix Biomedix, Inc. Short bio-active peptides for cellular and immunological modulation
CN101878019A (en) * 2007-11-30 2010-11-03 赢创高施米特有限公司 Personal care and cosmetic composition containing tetrapeptides with the motifs gx1x2g, px1x2p, or px1x2k
CN108220315A (en) * 2016-12-22 2018-06-29 珠海冀百康生物科技有限公司 The preparation method and fusion protein of a kind of small molecular protein or polypeptide
CN107652355A (en) * 2017-10-26 2018-02-02 陕西慧康生物科技有限责任公司 The liquid-phase synthesis process of skin lightening peptide

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
PETER M. HWANG,ET AL.: "Targeted expression, purification, and cleavage of fusion proteins from inclusion bodies in Escherichia coli", 《FEBS LETTERS》 *
PETER M. HWANG,ET AL.: "Targeted expression, purification, and cleavage of fusion proteins from inclusion bodies in Escherichia coli", 《FEBS LETTERS》, vol. 588, 31 December 2014 (2014-12-31), pages 247 - 252, XP071253979, DOI: 10.1016/j.febslet.2013.09.028 *
周一江等: "人/鲑降钙素嵌合基因在大肠杆菌中的串联表达", 《中国海洋大学学报(自然科学版)》 *
周一江等: "人/鲑降钙素嵌合基因在大肠杆菌中的串联表达", 《中国海洋大学学报(自然科学版)》, vol. 37, no. 01, 30 January 2007 (2007-01-30), pages 103 *
张军等: "多肽串联基因构建策略", 《中国生物工程杂志》 *
张军等: "多肽串联基因构建策略", 《中国生物工程杂志》, vol. 29, no. 08, 15 August 2009 (2009-08-15), pages 107 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113616807A (en) * 2021-07-26 2021-11-09 中山大学 Mitochondrial-targeted polypeptide and preparation method and application thereof
CN113616807B (en) * 2021-07-26 2023-07-21 中山大学 Mitochondrion-targeted polypeptide and preparation method and application thereof

Also Published As

Publication number Publication date
CN112759653B (en) 2023-06-23

Similar Documents

Publication Publication Date Title
JP2000504574A (en) Recombinant preparation of calcitonin fragments and its use in the preparation of calcitonin and related analogs
Liew et al. Preparation of recombinant thioredoxin fused N-terminal proCNP: analysis of enterokinase cleavage products reveals new enterokinase cleavage sites
CN111117977B (en) Recombinant polypeptide linked zymogen, preparation method, activation method and application thereof
US10000544B2 (en) Process for production of insulin and insulin analogues
CN112739823A (en) Pyrrolysinyl-tRNA synthetases
KR20180130452A (en) Mass production technology of recombinant botulinum toxin protein
KR101435606B1 (en) Method for in vivo residue-specific DOPA incorporation into mussel adhesive proteins
CN116622795A (en) Preparation method of L-amino acid ligase and application of L-amino acid ligase in dipeptide synthesis
KR20080039879A (en) Method for preparing soluble and active recombinant proteins using pdi as a fusion partner
CN113201074B (en) PKEK fusion protein and preparation method and application thereof
CN112759653B (en) PKEK fusion protein and preparation method and application thereof
CN111499759A (en) Zinc finger protein-lactoferrin fusion protein with cell membrane penetrating property and preparation and application thereof
JP2023531168A (en) Insulin degludec derivative and its production method and use
JP7266325B2 (en) Fusion proteins containing fluorescent protein fragments and uses thereof
CN109971776B (en) Protein purification method based on photocleavage motif
JP2010519894A (en) Protein expression in E. coli
CN101235084A (en) Method for preparing bone morphogenic protein BMP-2 mature peptide
WO2021170754A1 (en) Recombinant microalgae able to produce kttks peptides, polypeptides, or proteins and their derivatives and associated method and uses thereof
RU2453604C1 (en) Hybrid protein (versions), bacterial strain escherichia coli - hybrid protein producer (versions) and method for producing methionine-free human interferon alpha-2
WO2021170849A1 (en) Recombinant microalgae able to produce peptides, polypeptides or proteins of collagen, elastin and their derivatives in the chloroplast of microalgae and associated method thereof
RU2441072C1 (en) FUSION PROTEIN, ESCHERICHIA COLI STRAIN BEING FUSION PROTEIN PRODUCER AND METHOD FOR PRODUCING METHIONINE-FREE HUMAN INTERFERON ALPHA-2b OF SUCH FUSION PROTEIN
US20160009779A1 (en) Novel fusion tags and expression vector system for the expression of human parathyroid hormone (rhpth)
CN117801123B (en) Wo Suoli peptide soluble intermediate, intermediate preparation method and Wo Suoli peptide preparation method
WO2012048856A1 (en) Proinsulin with helper sequence
CN107217069B (en) Prokaryotic expression vector, rbFGF-2 expression method, engineering bacteria and application

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant