CN113201074A - PKEK fusion protein, preparation method and application - Google Patents

PKEK fusion protein, preparation method and application Download PDF

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CN113201074A
CN113201074A CN202110494341.9A CN202110494341A CN113201074A CN 113201074 A CN113201074 A CN 113201074A CN 202110494341 A CN202110494341 A CN 202110494341A CN 113201074 A CN113201074 A CN 113201074A
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ala
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pkek
pro
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CN113201074B (en
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杨晓纯
曹春来
刘合栋
李素雯
李张万金
梁雄基
樊昌
梁芷瑜
周翠
何秀仪
黄国周
郭凤羽
张鹏
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Zhuhai United Laboratories Co Ltd
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Abstract

The invention discloses a PKEK fusion protein, a preparation method and application thereof. The structure formula of the PKEK fusion protein is as follows: (PKEK-B) n; b is flexible connecting peptide; n is an integer of 1 to 30. The fusion protein is expressed in the form of inclusion body protein, and meanwhile, a plurality of whitening polypeptides PKEK and flexible connecting peptide 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 polypeptides PKEK can be efficiently obtained, and the purity can reach 95.4%; in addition, the preparation method for producing the whitening polypeptide PEKE by using the genetic engineering technology can obtain a large amount of PKEK fusion protein inclusion bodies within 12 hours, the expression amount can reach 45g/L at most, the process is simple and efficient, the amplification is easy, and the industrial production is facilitated.

Description

PKEK fusion protein, preparation method and application
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
In the patent applications W0/2008/08M94 and W0/2009/068351, a novel whitening polypeptide PKEK is described, 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 women in the Caucasian area use the PKEK and the vitamin C sodium phosphate together for 6 weeks, facial spots obviously fade, but the spots fade obviously when the PKEK or the vitamin C sodium phosphate are used 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.
The patent application 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 a 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 application CN106560475A discloses a genetic engineering biosynthesis process of active short peptides, which comprises connecting active short peptides in series, expressing the active short peptides and purification tags in microbial cells as soluble proteins or inclusion body proteins, and degrading recombinant proteins with high-specificity protease to finally form single active short peptides. 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, lysine side chain amino groups in free PKEK series-connected polypeptides are easy to be acetylated and modified, and the method cannot solve the problem of miscut of the PKEK series-connected polypeptides in an 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 simultaneously, the whitening polypeptide PKEK and flexible connecting peptide 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.
Another object of the present invention is to provide a gene encoding the above PKEK fusion protein.
The invention also aims to provide the application of the coding gene of the 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 whitening polypeptides PKEK and flexible connecting peptides in series, and the structural formula of the protein is as follows: (PKEK-B) n; wherein: b is flexible connecting peptide; n is an integer of 1 to 30.
The structural formula of the PKEK fusion protein is preferably as follows: a to Dd- (PKEK-B) n; wherein: a is chaperonin; d is a protease enzyme cutting site; b is flexible connecting peptide; n is an integer of 1 to 30; d is an integer of 0 to 10.
N is preferably 10-30; more preferably 10 to 20.
D is preferably 1-5; more preferably 1.
The chaperone protein is a combination formed by one or more than two of KSI, PagP, PaP3.30, TAF12, GDF15, GYGB, mKSI, mPGP, mPAP3.30, mGDF15, mGYGB and mTAF12, and m represents a mutant. The chaperonin has the functions of assisting the fusion protein to form an inclusion body in escherichia coli, promoting the formation of the inclusion body, improving the yield and quality of the inclusion body 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, wild-type PaP3.30 protein fragment and wild-type GYGB 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 KSI, PagP, PaP3.30, GDF15, GYGB or TAF12 proteins are selected as chaperone proteins; in addition, the wild-type chaperone protein has a plurality of lysine residues and arginine residues, so that excessive mistaken enzyme cutting fragments can be formed in a subsequent enzyme cutting process, and 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 can be remarkably improved, so that the chaperone protein can also be a mutant of KSI, PagP, PaP3.30, GDF15, GYGB or TAF12 protein, namely mKSI, mPGP, mPAP3.30, mGDF15, mGYGB or mTAF 12. The chaperone protein is a single molecular chaperone or a combination of molecular chaperones as described above, preferably Pagp + Pagp, KSI + KSI, Pagp + KSI, KSI + Pagp, TAF12+ KSI, KSI + TAF12, TAF12+ Pagp, TAF12+ GYGB or Pagp + GYGB; more preferably KSI + Pagp, TAF12+ KSI, TAF12+ GYGB or TAF12+ Pagp.
The flexible connecting peptide is Xm---X2-X1A peptide sequence as shown; wherein, X1As protease cleavage site, X2To XmIs a flexible segment; m is an integer of 0-10, m is 0 and represents the deletion of the flexible connecting peptide, m is 1, namely the flexible connecting peptide is only 1 amino acid, and the like; preferably, when m is 0, d cannot be 0, and n is 1-30; or when m is 0 and d is 0, n cannot be 1.
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.
M is preferably an integer of 1-9; more preferably an integer of 2 to 6.
The flexible connecting peptide is preferably AK, GK, AAK, AAGK, AAGGK, GGAAK, GGSASK or AAGGSGAGK.
When the chaperonin is TAF12 or chaperonin formed by fusing TAF12 with other chaperonin, the flexible connecting peptide is preferably AK, GK, AAK, AAGK, AAGGK or GGAAK; more preferably GK, AAK, AAGK or GGAAK; most preferably AAK, AAGK or GGAAK; the n is preferably 10-30; more preferably 10 to 20.
The other chaperone protein is preferably Pagp, KSI or GYGB.
When the chaperonin is KSI or chaperonin formed by KSI and other chaperonin fusion, the flexible connecting peptide is preferably AK, GK, AAK, AAGK, AAGGK or GGAAK; more preferably GGAAK or GGSASK; the n is preferably 10-20.
The other chaperone protein is preferably Pagp or TAF 12.
When the chaperonin is PagP or chaperonin formed by fusion of PagP and other chaperonin, the flexible connecting peptide is preferably AK, GK, AAK, AAGK, AAGGK, GGAAK or GGSASK; more preferably AAK; the n is preferably 10-20.
Preferably, the other chaperone protein is KSI or TAF 12.
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.4, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.31, SEQ ID NO.33, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.44 or SEQ ID NO. 52; preferably a sequence as shown in SEQ ID NO.4, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.33, SEQ ID NO.36, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.42 or SEQ ID NO. 52.
A recombinant vector comprising the gene encoding the PKEK fusion protein; the gene encoding the PKEK fusion protein is cloned into an expression vector to obtain the PKEK fusion protein.
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-29a (+), pET-28a (+), pET-22b (+), pET-26b (+), or pET-31b (+); most preferably pET-31b (+) or pET29a (+).
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 preferably comprises the following specific 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 the recombinant vector containing the PKEK fusion protein coding gene, wherein the structure of the coded fusion protein is A-Dd- (PKEK-B) n; wherein: a is chaperonin; b is a flexible linker peptide, such as Xm---X2-X1The peptide sequence shown, X1As protease cleavage site, X2To XmIs a flexible segment, and m is an integer of 0-10; d is a protease cleavage site; n is an integer of 1 to 30; d is an integer of 0 to 10;
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, and the recombinant expression vector containing the whitening polypeptide is introduced into the genetic engineering strain, so that 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 a nitrogen source, a carbon source, and inorganic salts.
The nitrogen source is one or two of an organic nitrogen source and an inorganic nitrogen source.
The composition of the fermentation medium is preferably as follows: 2.5-3.5 g/L yeast powder, 4-6 g/L, NaCl 0.5.5-1.5 g/L, KH g peptone2PO4 2.5～3.5g/L、Na2HPO4 3～3.5g/L、MgSO4 0.5～1.5g/L、(NH4)2 SO 4 5～7g/L、CaCl2·2H20.01-0.015 g/L of O, 4-4.2 g/L of glycerol, 0.8-1 mL/L of trace elements and water as a solvent; adjusting the pH value to 6.8-7.2 by using ammonia water after sterilization; more preferably as follows: 3g/L yeast powder, 5g/L, NaCl 1g/L, KH g peptone2PO4 3g/L、Na2HPO4 3.25g/L、MgSO4 1g/L、(NH4)2SO4 6g/L、CaCl2·2H2O0.01375 g/L, glycerol 4.125g/L, trace elements 0.875mL/L, and water as solvent; after sterilization, the pH was adjusted to 7.0 with ammonia.
The composition of the trace element mixed solution (total volume 100mL) is as follows: FeCl2·4H2O 2～2.5g、ZnCl20.1～0.15g、 CoCl2·6H2O 0.15～0.25g、Na2Mo4·2H2O 0.15～0.25g、CaCl2·2H2O 0.05～0.15g、CuCl2·2H2O 0.1～ 0.15g、H3BO4 0.03～0.06g、MnSO4·H20.2-0.23 g of O, 9.5-10.5 mL of 37 wt% concentrated hydrochloric acid and water as a solvent; the following are preferred: FeCl2·4H2O 2.287g、ZnCl2 0.131g、CoCl2·6H2O 0.2g、Na2Mo4·2H2O 0.2g、CaCl2·2H2O 0.1g、CuCl2·2H2O 0.125g、H3BO4 0.05g、MnSO4·H2O0.217 g, concentrated hydrochloric acid 10mL of 37 wt%, and water as a solvent.
The fermentation mode is high-density fermentation.
The specific steps of the fermentation are preferably as follows: fermenting at 36-38 deg.C, pH controlled at 6.8-7.2, and maximum oxygen saturation of Dissolved Oxygen (DO) 5-50%; feeding was started when the culture was completed to carbon source depletion and the culture was completed to the OD600And when the concentration is 75-85 ℃, adding IPTG for induction.
The feed medium used for feeding comprises the following components: 450-550 g/L of glycerol, 30-40 g/L of yeast extract powder, 60-70 g/L of yeast peptone, 3-4 mL/L of trace elements and the balance of water; the preferred composition is as follows: 500g/L of glycerol, 33g/L of yeast extract powder, 66/L g of yeast peptone, 3.5mL/L of trace elements and the balance of water.
The feeding rate is preferably 0.4 mL-min-1·L-1(ii) a More preferably 0.3 to 0.5 mL/min-1·L-1。
The OD600Is 80.
The final concentration of the IPTG in a fermentation system is 0.2-0.4 mM; more preferably 0.3 mM.
The induction time is at least 3 h; more preferably 12 to 15 hours.
The inclusion body described in step S2 is obtained by disrupting the bacterial cells by a high-pressure homogenization method.
The inclusion body obtaining step described in step S2 is preferably as follows: carrying out solid-liquid separation on the fermentation liquor to obtain thalli; and (3) suspending the thalli in a crushing liquid, homogenizing and crushing the thalli in a water bath, centrifugally collecting the precipitate, dissolving the precipitate in a washing buffer solution for washing, and centrifugally collecting the inclusion body precipitate.
The solid-liquid separation is preferably performed by centrifugation.
The ratio of the cell to the disrupted solution is preferably 1 g: mixing in a ratio of 5-10 mL; more preferably, the amount of the compound is 1 g: mixing in a proportion of 7 mL.
The composition of the crushing liquid is preferably as follows: 6-6.2 g/L Tris and 1.4-1.5 g/L EDTA, and the balance of purified water, and adjusting the pH value to 7.8-8.2; more preferably as follows: 6.05g/L Tris and 1.46g/L EDTA, the balance being purified water, the pH being adjusted to 8.0.
The homogenization conditions are preferably 900 bar.
The precipitate and the washing buffer are preferably mixed in a ratio of 1 g: mixing 10-20 mL of the mixture; more preferably, the amount of the compound is 1 g: 15mL of the mixture is mixed.
The composition of the wash buffer is preferably as follows: 6-6.1 g/L Tris, 1.4-1.5 g/L EDTA, 2.8-3.0 g/L NaCl, 9-11 mL/L TritioX-100, and the balance of water; the following are preferred: 6.05g/L Tris, 1.46g/L EDTA, 2.9g/L NaCl, 10mL/L TritioX-100 and the balance of water.
The protease used in the enzyme digestion in step S3 is one or at least two of bovine trypsin, porcine trypsin and 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 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 calculated according to the proportion of 1g of the inclusion body to 5.5-6.5 mL of the urea solution; more preferably, the amount is calculated based on 1g of inclusion body in 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 inventor of the invention connects PKEK and flexible connecting peptide 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 simultaneously, the whitening polypeptide PKEK and flexible connecting peptide 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 can be efficiently obtained, and the purity can reach 95.4%; in addition, the preparation method for producing the whitening polypeptide PEKE by using the genetic engineering technology can obtain a large amount of PKEK fusion protein inclusion bodies within 12 hours, the expression amount can reach 45g/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-TAF12-K- (PKEK-AAK) described in example 110Map of the plasmid.
FIG. 2 shows the genetically engineered strain BL21(DE)/pET31b-TAF12-K- (PKEK-AAK) 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-TAF12-K- (PKEK-AAK) provided in example 110Performing shake flask fermentation and 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 1 is negative control, and Lanes 2-7 represent different monoclonals.
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 NdeI and XhoI were purchased from NEB; TAF12-K- (PKEK-AAK)10The fusion gene sequence (shown as SEQ ID NO. 4) is entrusted to Nanjing Kinshire company for synthesis; the agarose gel recovery kit is purchased from Shanghai GmbH of Biotechnology (bioengineering); DNA Ligation Kit was purchased from TaKaRa; escherichia coli DH5 α was purchased from Life Technologies; the plasmid extraction kit is purchased from Shanghai GmbH of Biotechnology (bioengineering); identification of proteins containing TAF12-K- (PKEK-AAK)10Cloning of the gene fragment sequencing was entrusted to Invitrogen corporation; 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 Suzhou, Nami, MicroTechnical, Inc.
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-TAF12-K- (PKEK-AAK)10
1.1 Artificial synthesis of Gene sequence TAF12-K- (PKEK-AAK) containing molecular chaperone, protease cleavage site, PKEK and connecting peptide10
Designing gene sequence TAF12-K- (PKEK-AAK) containing molecular chaperone, protease cutting site, PKEK and connecting peptide10The fusion gene fragment of (1) converts the amino acid sequences into nucleotide sequences according to a codon table, selects codons with higher use frequency according to codon use preference of escherichia coli in the conversion process, adjusts GC content of the codons, removes cis-acting elements and repetitive sequences influencing gene transcription to optimize the codons, introduces double-stop codons TAATGA at the 3' end of the gene sequence, and is convenient for gene operation and is positioned at TAF12-K- (PKEK-AAK)10The NdeI restriction site sequence CATATG is introduced into the 5' end of the fusion gene sequence and is positioned at TAF12-K- (PKEK-AAK)10XhoI enzyme cutting site CTCGAG is introduced into the 3' end of the fusion gene, and optimized TAF12-K- (PKEK-AAK)10The fusion gene sequence is shown in SEQ ID NO. 4.
1.2pET31b-TAF12-K-(PKEK-AAK)10Construction of recombinant expression vectors
Plasmid pET-31b (+) was double-digested with restriction enzymes NdeI and XhoI, 5. mu.L of the digested product was analyzed by agarose gel electrophoresis at a mass/volume ratio of 0.8%, after completion of the digestion, 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 5.3kb was cut, and the DNA fragment in the gel was purified into 30. mu.L of deionized water using an agarose gel recovery kit to obtain a plasmid fragment with a sticky end. Similarly, the NdeI and XhoI are used for treating TAF12-K- (PKEK-AAK)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. With DNA Ligation kit (DNA Ligation K)it) the gene fragment and the plasmid fragment were ligated at 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. 5);
t7 terminator primer: 5'-TGCTAGTTATTGCTCAGCGG-3' (SEQ ID NO. 6).
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 the clone of a gene fragment of about 465bp and a vector fragment of about 5.3kb, namely the clone containing TAF12-K- (PKEK-AAK)10Cloning of the gene fragment. Will identify the compounds containing TAF12-K- (PKEK-AAK)10The cloning of the gene fragment was sequenced. Inoculating the single clone without base mutation and frame shift proved by sequencing to 50mL LB liquid culture medium added with ampicillin (final concentration is 100 mug/mL), culturing at 37 deg.C and 250rpm for 18h, extracting the plasmid from the obtained culture with plasmid extractant box, namely recombinant expression vector, named as pET31b-TAF12-K- (PKEK-AAK)10As shown in fig. 1.
2、BL21(DE3)/pET31b-TAF12-K-(PKEK-AAK)10Screening of expression strains and product identification
According to Mole gram published by Cold spring harbor laboratory in the United statesColi BL21(DE3) competent cells were prepared by the calcium chloride method provided in the third edition of the Longe laboratory Manual. mu.L of the recombinant expression vector pET31b-TAF12-K- (PKEK-AAK) obtained in 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) and inverted at 37 ℃ until a single colony appeared, to obtain a strain library of PKEK fusion protein expression, which was named BL21(DE3)/pET31b-TAF12-K- (PKEK-AAK)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 LB solid culture medium by using sterile toothpicks, picking 6 single colonies, inoculating to 50mL LB liquid culture medium, carrying out shake culture at 37 ℃ and 250rpm, and obtaining the OD of bacterial liquid600When the concentration is 0.5-1.0, inoculating 3-6 mL of the bacterial liquid into 100mL of LB liquid medium, inoculating 3-6 mL of BL21(DE3)/pET31b bacterial liquid into 100mL of LB liquid medium as a negative control, and performing shaking culture at 37 ℃ and 250rpm until OD is reached600When the concentration is 0.6 to 0.8, IPTG (isopropyl-. beta. -D-thiogalactopyranoside, final concentration is 1mmol/L) is added to start induction, bacterial solutions (4mL) are taken for induction for 3h and 15h respectively, centrifuged at 12000rpm for 1 minute, the supernatant is removed, and the cells are stored 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 gray scale scanning by using a biological image processing software Gel-Pro Analyzer 4.0 as shown in figure 2 and figure 3 The ratio of the bands is the purity of the PKEK fusion protein in 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-TAF12-K- (PKEK-AAK)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-TAF12-K- (PKEK-AAK)10The protein with the size of about 17kDa can be expressed after IPTG induction, which is consistent with the theoretical molecular weight of 16.947KDa (obtained by using online software http:// web. expasy. org/computer _ pi/prediction) of PKEK fusion protein, and indicates that PKEK gene has been successfully introduced into the genetic engineering bacteria.
The genetically engineered bacterium BL21(DE3)/pET31b-TAF12-K- (PKEK-AAK)10Streaking on fresh LB solid medium with final concentration of 100. mu.g/mL ampicillin to perform pure culture, picking out single colony, and culturing with LB liquid medium to OD600Preparing a bacterial liquid containing 15% by volume of glycerol as 0.8-1.0, and storing the bacterial liquid in a refrigerator at the temperature of-70 ℃ for later use.
3. PKEK fusion protein gene engineering bacteria BL21(DE3)/pET31b-TAF12-K- (PKEK-AAK)10Fermentation test
3.1 preparation of seed cultures
Collecting 20 μ L of BL21(DE3)/pET31b-TAF12-K- (PKEK-AAK) frozen at-70 deg.C10The strain was inoculated into 50mL of LB liquid medium containing ampicillin (final concentration: 100. mu.g/mL) at 28 ℃,Culturing at 250rpm for 16 hr to activate strain, inoculating 50mL of activated strain into 400mL LB liquid medium, culturing at 28 deg.C and 250rpm for 3 hr to obtain seed culture, and controlling OD600Between 2.0 and 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 are cultured to a cell concentration OD600Approximately equal to 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 powder (Yeast Extract) 24.0
Peptone (Tryptone) 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 purified water by 10 times, and measuring TAF12-K- (PKEK-AAK)10Expression of the fusion protein (two parallel batches). Genetically engineered strain
BL21(DE)/pET31b-TAF12-K-(PKEK-AAK)10The average yield of inclusion bodies containing the fusion protein after conversion in the fermentation test was 45g/L (measured as inclusion bodies isolated in step 4.1 below), wherein the content of the fusion protein in the inclusion bodies of the fusion protein was 44%, i.e., 19.8 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 instruction, and obtaining a PKEK crude product by enzyme digestion.
The liquid phase detection conditions of the PKEK crude product are as follows: BEH-C8 (filler size 1.7 μm, column size 2.1X 100mm) analyzed the column at a column temperature of 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%. The PKEK peak-off time is 21.5min, the statistical peak area ratio is 13.2%, namely the purity of the PKEK crude product is 13.2%.
5. Purifying the enzyme-digested PKEK crude product
100mL of the crude cleaved PKEK prepared as described above in step 4 was loaded onto a chromatography column packed with 10mL of Uni PMM30-500 packing (which was previously equilibrated with a buffer containing 0.1% (v/v) TFA in 20% isopropanol) and after loading was completed, washed with equilibration buffer (a buffer containing 0.1% (v/v) TFA in 20% isopropanol) until baseline plateaus. Then, the column was eluted 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) at an elution flow rate of 2 column volumes per hour or less. And (3) collecting main peaks in sections, starting to collect about 20% of peak heights, and obtaining the PKEK with the detection wavelength of 280 nm. The purity of the PKEK prepared by the purification process reaches 95.4 percent. PKEK liquid phase detection conditions: BEH-C8 (filler size 1.7 μm, column size 2.1X 100mm) analyzed the column at a column temperature of 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 Seimerfer high-resolution liquid chromatograph-mass spectrometer for high-resolution mass spectrometry, deconvoluting by using a Thermo BioPharmaFinder2.0, and collecting the range: MS 100-1000. The result shows that the molecular weight of the prepared PKEK is 500.3Da which 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 PKEK can be obtained by enzyme digestion, and the purity of the crude product of PKEK is 13.2%.
Example 3
The method of this example is substantially the same as that described in 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 PKEK can be obtained by enzyme digestion, and the purity of the crude product of PKEK is 13.5%.
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 cleaved PKEK are shown in table 4.
Table 3: fermentation yield test of fusion proteins of different molecular chaperones and connecting peptides
Figure BDA0003053671650000071
Description of the drawings: the relative yields in the above table refer only to the relative yields of the PKEK portion of the fusion protein molecule, from which chaperones, protease sites and linker peptides have been subtracted.
In the above table, the nucleotide cleavage sites of the protein molecules are NdeI and XhoI. The amino acid sequence of the KSI molecular chaperone is shown as SEQ ID NO. 29; the amino acid sequence of the PagP molecular chaperone is shown as SEQ ID NO. 24; the amino acid sequence of the molecular chaperone TAF12 is shown in SEQ ID NO. 1; the amino acid sequence of the PaP3.30 molecular chaperone is shown as SEQ ID NO. 45; the amino acid sequence of the GDF15 molecular chaperone is shown in SEQ ID NO. 48; the amino acid sequence of the GYGB molecular chaperone is shown in SEQ ID NO. 34. The recombinant expression vector used in SEQ ID NO.4 was prepared by example 1. In the invention, the 5 'end of all the nucleotide sequences is enzyme cutting site NdeI, the 3' end is stop codon (taatga) and enzyme cutting site XhoI, and the first amino acid from the beginning of the amino acid sequence is M which comes from the enzyme cutting site NdeI. The results in table 3 show that the PKEK yield increases significantly with the addition of chaperones. Co-expression of certain hydrophobic chaperones may further increase PKEK production.
The different inclusion bodies were digested as in example 1, and the relative purity of the digested PKEK monomers is shown in table 4.
Table 4: PKEK relative purity of different molecular chaperones and connecting peptide fusion proteins after enzyme digestion
Molecular chaperones Protease cleavage site Linker peptide Number of PKEK-linker peptides in tandem Relative purity of PKEK monomers
TAF12 K Is free of 10 38％
TAF12 K Is free of 20 40％
TAF12 K AAK 10 100％
TAF12 K AK 10 99％
TAF12 K GK 10 106％
TAF12 K AAGK 10 110％
TAF12 K GGAAK 10 155％
TAF12 K WTK 10 39％
TAF12 K PPK 10 35％
TAF12+Pagp K AAK 10 100％
TAF12+Pagp K AAK 20 106％
TAF12+KSI K AAK 10 88％
TAF12+KSI K AAK 20 92％
TAF12+GYGB K AAK 30 109％
KSI K GGAAK 10 96％
KSI K GGSASK 20 104％
Pagp K AAK 10 103％
KSI+Pagp K AAK 10 91％
Pap3.30 K AAK 10 87％
GDF15 K AAK 10 95％
GYGB K AAK 10 108％
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 percentage indicates the relative purity after digestion, and the higher the purity, the lower the proportion of miscut.
The results in table 4 illustrate that: in the absence of the linker peptide, the monomeric PKEK obtained by enzymatic cleavage is much lower than that of the molecule with the linker peptide. While, when X in the peptide is linkedm---X2When the polypeptide is small side chain hydrophobic flexible polypeptide such as A, G, AA, AAG, 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
<160> 56
<210> 1
<223> TAF12
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
65 70 75
<210> 2
<223> (PKEK- AAK) 10
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
1 5 10 15
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
20 25 30
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
35 40 45
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
50 55 60
Lys Glu Lys Ala Ala Lys
65 70
<210> 3
<223> TAF12-K-(PKEK- AAK) 10
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 Lys
65 70 75 80
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
85 90 95
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
100 105 110
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
115 120 125
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
130 135 140
Lys Glu Lys Ala Ala Lys
145 150
<210> 4
<223> TAF12-K-(PKEK- AAK) 10
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
aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct 300
gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa 360
gctgctaaac cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa 420
aaagctgcta aaccgaaaga aaaagctgct aaataatgac tcgag 465
<210> 5
<223> T7 promoter primer
taatacgact cactataggg 20
<210> 6
<223> T7 terminator primer
tgctagttat tgctcagcgg 20
<210> 7
<223> (PKEK)10
Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys
1 5 10 15
Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys
20 25 30
Pro Lys Glu Lys Pro Lys Glu Lys
35 40
<210> 8
<223> (PKEK)10
catatgccga aagaaaaacc gaaagaaaaa ccgaaagaaa aaccgaaaga aaaaccgaaa 60
gaaaaaccga aagaaaaacc gaaagaaaaa ccgaaagaaa aaccgaaaga aaaaccgaaa 120
gaaaaataat gactcgag 138
<210> 9
<223> (PKEK-AAK)10
catatgccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa 60
gctgctaaac cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa 120
aaagctgcta aaccgaaaga aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa 180
gaaaaagctg ctaaaccgaa agaaaaagct gctaaataat gactcgag 228
<210> 10
<223> (PKEK-AAK)20
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
1 5 10 15
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
20 25 30
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
35 40 45
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
50 55 60
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
65 70 75 80
Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala
85 90 95
Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
100 105 110
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
115 120 125
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
130 135 140
<210> 11
<223> (PKEK-AAK)20
catatgccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa 60
gctgctaaac cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa 120
aaagctgcta aaccgaaaga aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa 180
gaaaaagctg ctaaaccgaa agaaaaagct gctaaaccga aagaaaaagc tgctaaaccg 240
aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac cgaaagaaaa agctgctaaa 300
ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga aaaagctgct 360
aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct 420
gctaaataat gactcgag 438
<210> 12
<223> TAF12-K-(PKEK-AK) 10
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 Lys
65 70 75 80
Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys
85 90 95
Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys
100 105 110
Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys
115 120 125
Pro Lys Glu Lys Ala Lys Pro Lys Glu Lys Ala Lys
130 135 140
<210> 13
<223> TAF12-K-(PKEK-AK) 10
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
aaaccgaaag aaaaagctaa accgaaagaa aaagctaaac cgaaagaaaa agctaaaccg 300
aaagaaaaag ctaaaccgaa agaaaaagct aaaccgaaag aaaaagctaa accgaaagaa 360
aaagctaaac cgaaagaaaa agctaaaccg aaagaaaaag ctaaaccgaa agaaaaagct 420
aaataatgac tcgag 435
<210> 14
<223> TAF12-K-(PKEK-GK) 10
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 Lys
65 70 75 80
Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys
85 90 95
Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys
100 105 110
Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys
115 120 125
Pro Lys Glu Lys Gly Lys Pro Lys Glu Lys Gly Lys
130 135 140
<210> 15
<223> TAF12-K-(PKEK-GK) 10
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
aaaccgaaag aaaaaggtaa accgaaagaa aaaggtaaac cgaaagaaaa aggtaaaccg 300
aaagaaaaag gtaaaccgaa agaaaaaggt aaaccgaaag aaaaaggtaa accgaaagaa 360
aaaggtaaac cgaaagaaaa aggtaaaccg aaagaaaaag gtaaaccgaa agaaaaaggt 420
aaataatgac tcgag 435
<210> 16
<223> TAF12-K-(PKEK-AAGK) 10
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 Lys
65 70 75 80
Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Lys
85 90 95
Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Lys
100 105 110
Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Lys
115 120 125
Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Lys
130 135 140
Pro Lys Glu Lys Ala Ala Gly Lys Pro Lys Glu Lys Ala Ala Gly Lys
145 150 155 160
<210> 17
<223> TAF12-K-(PKEK-AAGK) 10
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
aaaccgaaag aaaaagctgc tggtaaaccg aaagaaaaag ctgctggtaa accgaaagaa 300
aaagctgctg gtaaaccgaa agaaaaagct gctggtaaac cgaaagaaaa agctgctggt 360
aaaccgaaag aaaaagctgc tggtaaaccg aaagaaaaag ctgctggtaa accgaaagaa 420
aaagctgctg gtaaaccgaa agaaaaagct gctggtaaac cgaaagaaaa agctgctggt 480
aaataatgac tcgag 495
<210> 18
<223> TAF12-K-(PKEK-GGAAK) 10
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 Lys
65 70 75 80
Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala
85 90 95
Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly
100 105 110
Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu
115 120 125
Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro
130 135 140
Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala
145 150 155 160
Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys
165 170
<210> 19
<223> TAF12-K-(PKEK-GGAAK) 10
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
aaaccgaaag aaaaaggtgg tgctgctaaa ccgaaagaaa aaggtggtgc tgctaaaccg 300
aaagaaaaag gtggtgctgc taaaccgaaa gaaaaaggtg gtgctgctaa accgaaagaa 360
aaaggtggtg ctgctaaacc gaaagaaaaa ggtggtgctg ctaaaccgaa agaaaaaggt 420
ggtgctgcta aaccgaaaga aaaaggtggt gctgctaaac cgaaagaaaa aggtggtgct 480
gctaaaccga aagaaaaagg tggtgctgct aaataatgac tcgag 525
<210> 20
<223> TAF12-K-(PKEK-WTK) 10
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 Lys
65 70 75 80
Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys
85 90 95
Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys
100 105 110
Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr
115 120 125
Lys Pro Lys Glu Lys Trp Thr Lys Pro Lys Glu Lys Trp Thr Lys Pro
130 135 140
Lys Glu Lys Trp Thr Lys
145 150
<210> 21
<223> TAF12-K-(PKEK-WTK) 10
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
aaaccgaaag aaaaatggac caaaccgaaa gaaaaatgga ccaaaccgaa agaaaaatgg 300
accaaaccga aagaaaaatg gaccaaaccg aaagaaaaat ggaccaaacc gaaagaaaaa 360
tggaccaaac cgaaagaaaa atggaccaaa ccgaaagaaa aatggaccaa accgaaagaa 420
aaatggacca aaccgaaaga aaaatggacc aaataatgac tcgag 465
<210> 22
<223> TAF12-K-(PKEK-PPK) 10
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 Lys
65 70 75 80
Pro Lys Glu Lys Pro Pro Lys Pro Lys Glu Lys Pro Pro Lys Pro Lys
85 90 95
Glu Lys Pro Pro Lys Pro Lys Glu Lys Pro Pro Lys Pro Lys Glu Lys
100 105 110
Pro Pro Lys Pro Lys Glu Lys Pro Pro Lys Pro Lys Glu Lys Pro Pro
115 120 125
Lys Pro Lys Glu Lys Pro Pro Lys Pro Lys Glu Lys Pro Pro Lys Pro
130 135 140
Lys Glu Lys Pro Pro Lys
145 150
<210> 23
<223> TAF12-K-(PKEK-PPK) 10
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
aaaccgaaag aaaaaccgcc gaaaccgaaa gaaaaaccgc cgaaaccgaa agaaaaaccg 300
ccgaaaccga aagaaaaacc gccgaaaccg aaagaaaaac cgccgaaacc gaaagaaaaa 360
ccgccgaaac cgaaagaaaa accgccgaaa ccgaaagaaa aaccgccgaa accgaaagaa 420
aaaccgccga aaccgaaaga aaaaccgccg aaataatgac tcgag 465
<210> 24
<223> PagP chaperones
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
<210> 25
<223> TAF12-Pagp-K-(PKEK-AAK) 10
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 Met
65 70 75 80
Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln Thr
85 90 95
Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr Trp
100 105 110
His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn Glu
115 120 125
Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys Gly
130 135 140
Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn Lys
145 150 155 160
Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro Leu
165 170 175
Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val Thr
180 185 190
Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro Leu
195 200 205
Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile Pro
210 215 220
Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe Gln
225 230 235 240
Phe Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
245 250 255
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
260 265 270
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
275 280 285
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
290 295 300
Lys Pro Lys Glu Lys Ala Ala Lys
305 310
<210> 26
<223> TAF12-Pagp-K-(PKEK-AAK) 10
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
atgaacgctg acgaatggat gaccaccttc cgtgaaaaca tcgctcagac ctggcagcag 300
ccggaacact acgacctgta catcccggct atcacctggc acgctcgttt cgcttacgac 360
aaagaaaaaa ccgaccgtta caacgaacgt ccgtggggtg gtggtttcgg tctgtctcgt 420
tgggacgaaa aaggtaactg gcacggtctg tacgctatgg ctttcaaaga ctcttggaac 480
aaatgggaac cgatcgctgg ttacggttgg gaatctacct ggcgtccgct ggctgacgaa 540
aacttccacc tgggtctggg tttcaccgct ggtgttaccg ctcgtgacaa ctggaactac 600
atcccgctgc cggttctgct gccgctggct tctgttggtt acggtccggt taccttccag 660
atgacctaca tcccgggtac ctacaacaac ggtaacgttt acttcgcttg gatgcgtttc 720
cagttcaaac cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa 780
aaagctgcta aaccgaaaga aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa 840
gaaaaagctg ctaaaccgaa agaaaaagct gctaaaccga aagaaaaagc tgctaaaccg 900
aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaat aatgactcga g 951
<210> 27
<223> TAF12-Pagp-K-(PKEK-AAK) 20
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 Met
65 70 75 80
Asn Ala Asp Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln Thr
85 90 95
Trp Gln Gln Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr Trp
100 105 110
His Ala Arg Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn Glu
115 120 125
Arg Pro Trp Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys Gly
130 135 140
Asn Trp His Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn Lys
145 150 155 160
Trp Glu Pro Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro Leu
165 170 175
Ala Asp Glu Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val Thr
180 185 190
Ala Arg Asp Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro Leu
195 200 205
Ala Ser Val Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile Pro
210 215 220
Gly Thr Tyr Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe Gln
225 230 235 240
Phe Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
245 250 255
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
260 265 270
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
275 280 285
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
290 295 300
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
305 310 315 320
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
325 330 335
Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala
340 345 350
Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
355 360 365
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
370 375 380
<210> 28
<223> TAF12-Pagp-K-(PKEK-AAK) 20
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
atgaacgctg acgaatggat gaccaccttc cgtgaaaaca tcgctcagac ctggcagcag 300
ccggaacact acgacctgta catcccggct atcacctggc acgctcgttt cgcttacgac 360
aaagaaaaaa ccgaccgtta caacgaacgt ccgtggggtg gtggtttcgg tctgtctcgt 420
tgggacgaaa aaggtaactg gcacggtctg tacgctatgg ctttcaaaga ctcttggaac 480
aaatgggaac cgatcgctgg ttacggttgg gaatctacct ggcgtccgct ggctgacgaa 540
aacttccacc tgggtctggg tttcaccgct ggtgttaccg ctcgtgacaa ctggaactac 600
atcccgctgc cggttctgct gccgctggct tctgttggtt acggtccggt taccttccag 660
atgacctaca tcccgggtac ctacaacaac ggtaacgttt acttcgcttg gatgcgtttc 720
cagttcaaac cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa 780
aaagctgcta aaccgaaaga aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa 840
gaaaaagctg ctaaaccgaa agaaaaagct gctaaaccga aagaaaaagc tgctaaaccg 900
aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac cgaaagaaaa agctgctaaa 960
ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga aaaagctgct 1020
aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct 1080
gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa 1140
gctgctaaat aatgactcga g 1161
<210> 29
<223> KSI chaperones
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
115 120
<210> 30
<223> TAF12-KSI-K-(PKEK-AAK) 10
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 Met
65 70 75 80
His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala Ala
85 90 95
Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp Asp
100 105 110
Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr Ala
115 120 125
Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala Val
130 135 140
Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe Ala
145 150 155 160
Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala Pro
165 170 175
Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile Arg
180 185 190
Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys 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
<210> 31
<223> TAF12-KSI-K-(PKEK-AAK) 10
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
atgcacaccc cggaacacat caccgctgtt gttcagcgtt tcgttgctgc tctgaacgct 300
ggtgacctgg acggtatcgt tgctctgttc gctgacgacg ctaccgttga agacccggtt 360
ggttctgaac cgcgttctgg taccgctgct atccgtgaat tctacgctaa ctctctgaaa 420
ctgccgctgg ctgttgaact gacccaggaa gttcgtgctg ttgctaacga agctgctttc 480
gctttcaccg tttctttcga ataccagggt cgtaaaaccg ttgttgctcc gatcgaccac 540
ttccgtttca acggtgctgg taaagttgtt tctatccgtg ctctgttcgg tgaaaaaaac 600
atccacgctt gcaaaccgaa agaaaaagct gctaaaccga aagaaaaagc tgctaaaccg 660
aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac cgaaagaaaa agctgctaaa 720
ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga aaaagctgct 780
aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaataatg actcgag 837
<210> 32
<223> TAF12-KSI-K-(PKEK-AAK) 20
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 Met
65 70 75 80
His Thr Pro Glu His Ile Thr Ala Val Val Gln Arg Phe Val Ala Ala
85 90 95
Leu Asn Ala Gly Asp Leu Asp Gly Ile Val Ala Leu Phe Ala Asp Asp
100 105 110
Ala Thr Val Glu Asp Pro Val Gly Ser Glu Pro Arg Ser Gly Thr Ala
115 120 125
Ala Ile Arg Glu Phe Tyr Ala Asn Ser Leu Lys Leu Pro Leu Ala Val
130 135 140
Glu Leu Thr Gln Glu Val Arg Ala Val Ala Asn Glu Ala Ala Phe Ala
145 150 155 160
Phe Thr Val Ser Phe Glu Tyr Gln Gly Arg Lys Thr Val Val Ala Pro
165 170 175
Ile Asp His Phe Arg Phe Asn Gly Ala Gly Lys Val Val Ser Ile Arg
180 185 190
Ala Leu Phe Gly Glu Lys Asn Ile His Ala Cys 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
340
<210> 33
<223> TAF12-KSI-K-(PKEK-AAK) 20
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
atgcacaccc cggaacacat caccgctgtt gttcagcgtt tcgttgctgc tctgaacgct 300
ggtgacctgg acggtatcgt tgctctgttc gctgacgacg ctaccgttga agacccggtt 360
ggttctgaac cgcgttctgg taccgctgct atccgtgaat tctacgctaa ctctctgaaa 420
ctgccgctgg ctgttgaact gacccaggaa gttcgtgctg ttgctaacga agctgctttc 480
gctttcaccg tttctttcga ataccagggt cgtaaaaccg ttgttgctcc gatcgaccac 540
ttccgtttca acggtgctgg taaagttgtt tctatccgtg ctctgttcgg tgaaaaaaac 600
atccacgctt gcaaaccgaa agaaaaagct gctaaaccga aagaaaaagc tgctaaaccg 660
aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac cgaaagaaaa agctgctaaa 720
ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga aaaagctgct 780
aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct 840
gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa 900
gctgctaaac cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa 960
aaagctgcta aaccgaaaga aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa 1020
gaaaaagctg ctaaataatg actcgag 1047
<210> 34
<223> GYGB
Met Glu Lys Val Pro Gly Glu Met Glu Ile Glu Arg Arg Glu Arg Ser
1 5 10 15
Glu Glu Leu Ser Glu Ala Glu Arg Lys Ala Val Gln Ala Met Trp Ala
20 25 30
Arg Leu Tyr Ala Asn Cys Glu Asp Val Gly Val Ala Ile Leu Val Arg
35 40 45
Phe Phe Val Asn Phe Pro Ser Ala Lys Gln Tyr Phe Ser Gln Phe Lys
50 55 60
His Met Glu Asp Pro Leu Glu Met Glu Arg Ser Pro Gln Leu Arg Lys
65 70 75 80
His Ala Cys Arg Val Met Gly Ala Leu Asn Thr Val Val Glu Asn Leu
85 90 95
His Asp Pro Asp
100
<210> 35
<211> 390
<223> TAF12-GYGB-K-(PKEK-AAK) 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 Met
65 70 75 80
Glu Lys Val Pro Gly Glu Met Glu Ile Glu Arg Arg Glu Arg Ser Glu
85 90 95
Glu Leu Ser Glu Ala Glu Arg Lys Ala Val Gln Ala Met Trp Ala Arg
100 105 110
Leu Tyr Ala Asn Cys Glu Asp Val Gly Val Ala Ile Leu Val Arg Phe
115 120 125
Phe Val Asn Phe Pro Ser Ala Lys Gln Tyr Phe Ser Gln Phe Lys His
130 135 140
Met Glu Asp Pro Leu Glu Met Glu Arg Ser Pro Gln Leu Arg Lys His
145 150 155 160
Ala Cys Arg Val Met Gly Ala Leu Asn Thr Val Val Glu Asn Leu His
165 170 175
Asp Pro Asp Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala
180 185 190
Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
195 200 205
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
210 215 220
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
225 230 235 240
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
245 250 255
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
260 265 270
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
275 280 285
Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala
290 295 300
Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
305 310 315 320
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
325 330 335
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
340 345 350
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
355 360 365
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
370 375 380
Lys Glu Lys Ala Ala Lys
385 390
<210> 36
<223> TAF12-GYGB-K-(PKEK-AAK) 30
catatgtccc cggaaaacaa ccaggtgctg accaaaaaaa aactgcagga cctggtgcgc 60
gaagtggacc cgaacgaaca gctggacgaa gacgtggaag aaatgctgct gcagatcgcc 120
gacgacttca tcgaatccgt ggtgaccgcc gcctgccagc tggcccgcca ccgcaaatcc 180
tccaccctgg aagtgaaaga cgtgcagctg cacctggaac gccagtggaa catgtggatc 240
atggaaaaag tgccgggcga aatggaaatc gaacgccgcg aacgctccga agaactgtcc 300
gaagccgaac gcaaagccgt gcaggccatg tgggcccgcc tgtacgccaa ctgcgaagac 360
gtgggcgtgg ccatcctggt gcgcttcttc gtgaacttcc cgtccgccaa acagtacttc 420
tcccagttca aacacatgga agacccgctg gaaatggaac gctccccgca gctgcgcaaa 480
cacgcctgcc gcgtgatggg cgccctgaac accgtggtgg aaaacctgca cgacccggac 540
aaaccgaaag aaaaagccgc caaaccgaaa gaaaaagccg ccaaaccgaa agaaaaagcc 600
gccaaaccga aagaaaaagc cgccaaaccg aaagaaaaag ccgccaaacc gaaagaaaaa 660
gccgccaaac cgaaagaaaa agccgccaaa ccgaaagaaa aagccgccaa accgaaagaa 720
aaagccgcca aaccgaaaga aaaagccgcc aaaccgaaag aaaaagccgc caaaccgaaa 780
gaaaaagccg ccaaaccgaa agaaaaagcc gccaaaccga aagaaaaagc cgccaaaccg 840
aaagaaaaag ccgccaaacc gaaagaaaaa gccgccaaac cgaaagaaaa agccgccaaa 900
ccgaaagaaa aagccgccaa accgaaagaa aaagccgcca aaccgaaaga aaaagccgcc 960
aaaccgaaag aaaaagccgc caaaccgaaa gaaaaagccg ccaaaccgaa agaaaaagcc 1020
gccaaaccga aagaaaaagc cgccaaaccg aaagaaaaag ccgccaaacc gaaagaaaaa 1080
gccgccaaac cgaaagaaaa agccgccaaa ccgaaagaaa aagccgccaa accgaaagaa 1140
aaagccgcca aaccgaaaga aaaagccgcc aaataatgac tcgag 1185
<210> 37
<211> 215
<223> KSI-K-(PKEK-GGAAK) 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 Lys Pro Lys Glu
115 120 125
Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala Lys Pro
130 135 140
Lys Glu Lys Gly Gly Ala Ala Lys Pro Lys Glu Lys Gly Gly Ala Ala
145 150 155 160
Lys Pro Lys Glu Lys 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
210 215
<210> 38
<223> KSI-K-(PKEK-GGAAK) 10
catatgcaca ccccggaaca catcaccgct gttgttcagc gtttcgttgc tgctctgaac 60
gctggtgacc tggacggtat cgttgctctg ttcgctgacg acgctaccgt tgaagacccg 120
gttggttctg aaccgcgttc tggtaccgct gctatccgtg aattctacgc taactctctg 180
aaactgccgc tggctgttga actgacccag gaagttcgtg ctgttgctaa cgaagctgct 240
ttcgctttca ccgtttcttt cgaataccag ggtcgtaaaa ccgttgttgc tccgatcgac 300
cacttccgtt tcaacggtgc tggtaaagtt gtttctatcc gtgctctgtt cggtgaaaaa 360
aacatccacg cttgcaaacc gaaagaaaaa ggtggtgctg ctaaaccgaa agaaaaaggt 420
ggtgctgcta aaccgaaaga aaaaggtggt gctgctaaac cgaaagaaaa aggtggtgct 480
gctaaaccga aagaaaaagg tggtgctgct aaaccgaaag aaaaaggtgg tgctgctaaa 540
ccgaaagaaa aaggtggtgc tgctaaaccg aaagaaaaag gtggtgctgc taaaccgaaa 600
gaaaaaggtg gtgctgctaa accgaaagaa aaaggtggtg ctgctaaata atgactcgag 660
<210> 39
<223> KSI-K-(PKEK-GGSASK) 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 Lys Pro Lys Glu
115 120 125
Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser
130 135 140
Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly
145 150 155 160
Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro
165 170 175
Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser
180 185 190
Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu
195 200 205
Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser
210 215 220
Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly
225 230 235 240
Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro
245 250 255
Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser
260 265 270
Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu
275 280 285
Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser
290 295 300
Lys Pro Lys Glu Lys Gly Gly Ser Ala Ser Lys Pro Lys Glu Lys Gly
305 310 315 320
Gly Ser Ala Ser Lys
325
<210> 40
<223> KSI-K-(PKEK-GGSASK) 20
catatgcaca ccccggaaca catcaccgct gttgttcagc gtttcgttgc tgctctgaac 60
gctggtgacc tggacggtat cgttgctctg ttcgctgacg acgctaccgt tgaagacccg 120
gttggttctg aaccgcgttc tggtaccgct gctatccgtg aattctacgc taactctctg 180
aaactgccgc tggctgttga actgacccag gaagttcgtg ctgttgctaa cgaagctgct 240
ttcgctttca ccgtttcttt cgaataccag ggtcgtaaaa ccgttgttgc tccgatcgac 300
cacttccgtt tcaacggtgc tggtaaagtt gtttctatcc gtgctctgtt cggtgaaaaa 360
aacatccacg cttgcaaacc gaaagaaaaa ggtggttctg cttctaaacc gaaagaaaaa 420
ggtggttctg cttctaaacc gaaagaaaaa ggtggttctg cttctaaacc gaaagaaaaa 480
ggtggttctg cttctaaacc gaaagaaaaa ggtggttctg cttctaaacc gaaagaaaaa 540
ggtggttctg cttctaaacc gaaagaaaaa ggtggttctg cttctaaacc gaaagaaaaa 600
ggtggttctg cttctaaacc gaaagaaaaa ggtggttctg cttctaaacc gaaagaaaaa 660
ggtggttctg cttctaaacc gaaagaaaaa ggtggttctg cttctaaacc gaaagaaaaa 720
ggtggttctg cttctaaacc gaaagaaaaa ggtggttctg cttctaaacc gaaagaaaaa 780
ggtggttctg cttctaaacc gaaagaaaaa ggtggttctg cttctaaacc gaaagaaaaa 840
ggtggttctg cttctaaacc gaaagaaaaa ggtggttctg cttctaaacc gaaagaaaaa 900
ggtggttctg cttctaaacc gaaagaaaaa ggtggttctg cttctaaacc gaaagaaaaa 960
ggtggttctg cttctaaata atgactcgag 990
<210> 41
<223> Pagp-K-(PKEK-AAK) 10
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 Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
165 170 175
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
180 185 190
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
195 200 205
Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala
210 215 220
Ala Lys Pro Lys Glu Lys Ala Ala Lys
225 230
<210> 42
<223> Pagp-K-(PKEK-AAK) 10
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
ttccagttca aaccgaaaga aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa 540
gaaaaagctg ctaaaccgaa agaaaaagct gctaaaccga aagaaaaagc tgctaaaccg 600
aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac cgaaagaaaa agctgctaaa 660
ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aataatgact cgag 714
<210> 43
<223> KSI-Pagp-K-(PKEK-AAK) 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 Met Asn Ala Asp
115 120 125
Glu Trp Met Thr Thr Phe Arg Glu Asn Ile Ala Gln Thr Trp Gln Gln
130 135 140
Pro Glu His Tyr Asp Leu Tyr Ile Pro Ala Ile Thr Trp His Ala Arg
145 150 155 160
Phe Ala Tyr Asp Lys Glu Lys Thr Asp Arg Tyr Asn Glu Arg Pro Trp
165 170 175
Gly Gly Gly Phe Gly Leu Ser Arg Trp Asp Glu Lys Gly Asn Trp His
180 185 190
Gly Leu Tyr Ala Met Ala Phe Lys Asp Ser Trp Asn Lys Trp Glu Pro
195 200 205
Ile Ala Gly Tyr Gly Trp Glu Ser Thr Trp Arg Pro Leu Ala Asp Glu
210 215 220
Asn Phe His Leu Gly Leu Gly Phe Thr Ala Gly Val Thr Ala Arg Asp
225 230 235 240
Asn Trp Asn Tyr Ile Pro Leu Pro Val Leu Leu Pro Leu Ala Ser Val
245 250 255
Gly Tyr Gly Pro Val Thr Phe Gln Met Thr Tyr Ile Pro Gly Thr Tyr
260 265 270
Asn Asn Gly Asn Val Tyr Phe Ala Trp Met Arg Phe Gln Phe 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 Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala
305 310 315 320
Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
325 330 335
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
340 345 350
Glu Lys Ala Ala Lys
355
<210> 44
<223> KSI-Pagp-K-(PKEK-AAK) 10
catatgcaca ccccggaaca catcaccgct gttgttcagc gtttcgttgc tgctctgaac 60
gctggtgacc tggacggtat cgttgctctg ttcgctgacg acgctaccgt tgaagacccg 120
gttggttctg aaccgcgttc tggtaccgct gctatccgtg aattctacgc taactctctg 180
aaactgccgc tggctgttga actgacccag gaagttcgtg ctgttgctaa cgaagctgct 240
ttcgctttca ccgtttcttt cgaataccag ggtcgtaaaa ccgttgttgc tccgatcgac 300
cacttccgtt tcaacggtgc tggtaaagtt gtttctatcc gtgctctgtt cggtgaaaaa 360
aacatccacg cttgcatgaa cgctgacgaa tggatgacca ccttccgtga aaacatcgct 420
cagacctggc agcagccgga acactacgac ctgtacatcc cggctatcac ctggcacgct 480
cgtttcgctt acgacaaaga aaaaaccgac cgttacaacg aacgtccgtg gggtggtggt 540
ttcggtctgt ctcgttggga cgaaaaaggt aactggcacg gtctgtacgc tatggctttc 600
aaagactctt ggaacaaatg ggaaccgatc gctggttacg gttgggaatc tacctggcgt 660
ccgctggctg acgaaaactt ccacctgggt ctgggtttca ccgctggtgt taccgctcgt 720
gacaactgga actacatccc gctgccggtt ctgctgccgc tggcttctgt tggttacggt 780
ccggttacct tccagatgac ctacatcccg ggtacctaca acaacggtaa cgtttacttc 840
gcttggatgc gtttccagtt caaaccgaaa gaaaaagctg ctaaaccgaa agaaaaagct 900
gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa 960
gctgctaaac cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa 1020
aaagctgcta aaccgaaaga aaaagctgct aaaccgaaag aaaaagctgc taaataatga 1080
ctcgag 1086
<210> 45
<223> PaP3.30 chaperones
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
<210> 46
<223> PaP3.30-K-(PKEK-AAK) 10
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 Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala
165 170 175
Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
180 185 190
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
195 200 205
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
210 215 220
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
225 230
<210> 47
<223> PaP3.30-K-(PKEK-AAK) 10
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 gtaaaccgaa agaaaaagct gctaaaccga aagaaaaagc tgctaaaccg 540
aaagaaaaag ctgctaaacc gaaagaaaaa gctgctaaac cgaaagaaaa agctgctaaa 600
ccgaaagaaa aagctgctaa accgaaagaa aaagctgcta aaccgaaaga aaaagctgct 660
aaaccgaaag aaaaagctgc taaaccgaaa gaaaaagctg ctaaataatg actcgag 717
<210> 48
<223> GDF15
Met Ala Arg Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys
1 5 10 15
Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp
20 25 30
Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala
35 40 45
Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr
50 55 60
Ser Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val
65 70 75 80
Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly
85 90 95
Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys
100 105 110
Ile
<210> 49
<223> GDF15-K-(PKEK-AAK) 10
Met Ala Arg Asn Gly Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys
1 5 10 15
Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp
20 25 30
Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala
35 40 45
Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr
50 55 60
Ser Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val
65 70 75 80
Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr Gly
85 90 95
Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp Cys His Cys
100 105 110
Ile Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
115 120 125
Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys
130 135 140
Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
145 150 155 160
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
165 170 175
Lys Pro Lys Glu Lys Ala Ala Lys
180
<210> 50
<223> GDF15-K-(PKEK-AAK) 10
catatggctc gtaacggtga ccactgcccg ctgggtccgg gtcgttgctg ccgtctgcac 60
accgttcgtg cttctctgga agacctgggt tgggctgact gggttctgtc tccgcgtgaa 120
gttcaggtta ccatgtgcat cggtgcttgc ccgtctcagt tccgtgctgc taacatgcac 180
gctcagatca aaacctctct gcaccgtctg aaaccggaca ccgttccggc tccgtgctgc 240
gttccggctt cttacaaccc gatggttctg atccagaaaa ccgacaccgg tgtttctctg 300
cagacctacg acgacctgct ggctaaagac tgccactgca tcaaaccgaa agaaaaagct 360
gctaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa 420
gctgctaaac cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa 480
aaagctgcta aaccgaaaga aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa 540
gaaaaagctg ctaaataatg actcgag 567
<210> 51
<223> GYGB-K-(PKEK-AAK) 10
Met Glu Lys Val Pro Gly Glu Met Glu Ile Glu Arg Arg Glu Arg Ser
1 5 10 15
Glu Glu Leu Ser Glu Ala Glu Arg Lys Ala Val Gln Ala Met Trp Ala
20 25 30
Arg Leu Tyr Ala Asn Cys Glu Asp Val Gly Val Ala Ile Leu Val Arg
35 40 45
Phe Phe Val Asn Phe Pro Ser Ala Lys Gln Tyr Phe Ser Gln Phe Lys
50 55 60
His Met Glu Asp Pro Leu Glu Met Glu Arg Ser Pro Gln Leu Arg Lys
65 70 75 80
His Ala Cys Arg Val Met Gly Ala Leu Asn Thr Val Val Glu Asn Leu
85 90 95
His Asp Pro Asp Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys
100 105 110
Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala
115 120 125
Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro
130 135 140
Lys Glu Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys Pro Lys Glu
145 150 155 160
Lys Ala Ala Lys Pro Lys Glu Lys Ala Ala Lys
165 170
<210> 52
<223> GYGB-K-(PKEK-AAK) 10
catatggaaa aagttccggg tgaaatggaa atcgaacgtc gtgaacgttc tgaagaactg 60
tctgaagctg aacgtaaagc tgttcaggct atgtgggctc gtctgtacgc taactgcgaa 120
gacgttggtg ttgctatcct ggttcgtttc ttcgttaact tcccgtctgc taaacagtac 180
ttctctcagt tcaaacacat ggaagacccg ctggaaatgg aacgttctcc gcagctgcgt 240
aaacacgctt gccgtgttat gggtgctctg aacaccgttg ttgaaaacct gcacgacccg 300
gacaaaccga aagaaaaagc tgctaaaccg aaagaaaaag ctgctaaacc gaaagaaaaa 360
gctgctaaac cgaaagaaaa agctgctaaa ccgaaagaaa aagctgctaa accgaaagaa 420
aaagctgcta aaccgaaaga aaaagctgct aaaccgaaag aaaaagctgc taaaccgaaa 480
gaaaaagctg ctaaaccgaa agaaaaagct gctaaataat gactcgag 528
<210> 53
<223> TAF12-K-(PKEK)10
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 Lys
65 70 75 80
Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys
85 90 95
Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys
100 105 110
Pro Lys Glu Lys Pro Lys Glu Lys
115 120
<210> 54
<223> TAF12-K-(PKEK)10
catatgtccc cggaaaacaa ccaggtcctg accaagaaga agctgcagga cctggtccgc 60
gaagtcgacc cgaacgaaca gctggacgaa gacgtcgaag aaatgctgct gcagatcgcc 120
gacgacttca tcgaatccgt cgtcaccgcc gcctgccagc tggcccgcca ccgcaagtcc 180
tccaccctgg aagtcaagga cgtccagctg cacctggaac gccagtggaa catgtggatc 240
aagccgaagg aaaagccgaa ggaaaagccg aaggaaaagc cgaaggaaaa gccgaaggaa 300
aagccgaagg aaaagccgaa ggaaaagccg aaggaaaagc cgaaggaaaa gccgaaggaa 360
aagtaatgac tcgag 375
<210> 55
<223> TAF12-K-(PKEK)20
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 Lys
65 70 75 80
Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys
85 90 95
Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys
100 105 110
Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu Lys Pro Lys Glu 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
<210> 56
<223> TAF12-K-(PKEK)20
catatgtccc cggaaaacaa ccaggtcctg accaagaaga agctgcagga cctggtccgc 60
gaagtcgacc cgaacgaaca gctggacgaa gacgtcgaag aaatgctgct gcagatcgcc 120
gacgacttca tcgaatccgt cgtcaccgcc gcctgccagc tggcccgcca ccgcaagtcc 180
tccaccctgg aagtcaagga cgtccagctg cacctggaac gccagtggaa catgtggatc 240
aagccgaagg aaaagccgaa ggaaaagccg aaggaaaagc cgaaggaaaa gccgaaggaa 300
aagccgaagg aaaagccgaa ggaaaagccg aaggaaaagc cgaaggaaaa gccgaaggaa 360
aagccgaagg aaaagccgaa ggaaaagccg aaggaaaagc cgaaggaaaa gccgaaggaa 420
aagccgaagg aaaagccgaa ggaaaagccg aaggaaaagc cgaaggaaaa gccgaaggaa 480
aagtaatgac tcgag 495

Claims (14)

1. A PKEK fusion protein, comprising: the skin whitening peptide-based protein is a protein formed by sequentially connecting a plurality of skin whitening polypeptides PKEK and flexible connecting peptides in series, and has the following structural formula: (PKEK-B) n; b is flexible connecting peptide; n is an integer of 1 to 30.
2. The PKEK fusion protein of claim 1, wherein: the structural formula of the PKEK fusion protein is as follows: a to Dd- (PKEK-B) n; wherein: a is chaperonin; d is a protease enzyme cutting site; b is flexible connecting peptide; n is an integer of 1 to 30; d is an integer of 0 to 10.
3. The PKEK fusion protein of claim 2, wherein:
the flexible connecting peptide is Xm---X2-X1The peptide sequence shown, X1As protease cleavage site, X2To XmIs a flexible segment, and m is an integer of 0-10;
the chaperone protein is a combination formed by one or more than two of KSI, PagP, PaP3.30, TAF12, GDF15, GYGB, mKSI, mPGP, mPAP3.30, mGDF15, mGYGB and mTAF12, and m represents a mutant.
4. The PKEK fusion protein of claim 3, wherein:
the protease restriction site is Arg or Lys;
when m is 0, d cannot be 0 or n cannot be 1;
the amino acid in the flexible fragment cannot be Pro, Thr or Trp.
5. The PKEK fusion protein of claim 4, wherein:
n is 10-30;
m is an integer of 2-9;
the amino acid in the flexible fragment is Gly, Ala or Ser;
d is 1-5.
6. The PKEK fusion protein according to any of claims 1 to 5, characterized in that:
the flexible connecting peptide is AK, GK, AAK, AAGK, AAGGK, GGAAK, GGSASK or AAGGSGAGK.
7. The PKEK fusion protein of claim 6, wherein:
when the chaperonin is TAF12 or chaperonin formed by fusion of TAF12 and other chaperonin, the flexible connecting peptide is AK, GK, AAK, AAGK, AAGGK or GGAAK;
when the chaperonin is KSI or chaperonin formed by the fusion of KSI and other chaperonin, the flexible connecting peptide is AK, GK, AAK, AAGK, AAGGK or GGAAK;
when the chaperonin is PagP or chaperonin formed by fusion of PagP and other chaperonin, the flexible connecting peptide is AK, GK, AAK, AAGK, AAGGK, GGAAK or GGSASK.
8. A gene encoding the PKEK fusion protein of any of claims 1-7.
9. A recombinant vector characterized by: comprising the gene of claim 8; is obtained by cloning the gene of claim 8 into an expression vector.
10. A recombinant expression strain characterized by: comprising the recombinant vector of claim 9.
11. Use of the PKEK fusion protein according to any one of claims 1 to 5 for the preparation of a whitening polypeptide PEKE.
12. A preparation method for producing whitening polypeptide PEKE by utilizing a genetic engineering technology is characterized by comprising the following steps:
s1, obtaining a recombinant vector comprising a gene encoding a PKEK fusion protein, the structure of the encoded fusion protein being for example A-Dd- (PKEK-B) n; wherein: a is chaperonin; b is a flexible linker peptide, such as Xm---X2-X1The peptide sequence shown, X1As protease cleavage site, X2To XmIs a flexible segment, and m is an integer of 0-10; d is a protease enzyme cutting site; n is an integer of 1 to 30; d is an integer of 0 to 10;
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.
13. The method for preparing PEKE by genetic engineering technology according to claim 12, wherein the PEKE is prepared by the following steps:
the genetic engineering strain in the step S2 is Escherichia coli;
the fermentation medium used in the fermentation had the following composition: 2.5-3.5 g/L yeast powder, 4-6 g/L, NaCl 0.5.5-1.5 g/L, KH g peptone2PO4 2.5～3.5g/L、Na2HPO4 3～3.5g/L、MgSO4 0.5～1.5g/L、(NH4)2SO4 5～7g/L、CaCl2·2H20.01-0.015 g/L of O, 4-4.2 g/L of glycerol, 0.8-1 mL/L of trace elements and water as a solvent; adjusting the pH value to 6.8-7.2 by using ammonia water after sterilization;
the microelement mixed solution comprises the following components: per 100mL of FeCl2·4H2O 2～2.5g、ZnCl2 0.1～0.15g、CoCl2·6H2O 0.15～0.25g、Na2Mo4·2H2O 0.15～0.25g、CaCl2·2H2O 0.05～0.15g、CuCl2·2H2O 0.1～0.15g、H3BO4 0.03～0.06g、MnSO4·H20.2-0.23 g of O, 9.5-10.5 mL of 37 wt% concentrated hydrochloric acid and water as a solvent;
the fermentation comprises the following specific steps: fermenting at 36-38 deg.C, pH controlled at 6.8-7.2, and maximum oxygen saturation of dissolved oxygen at 5-50%; feeding was started when the culture was completed to carbon source depletion and the culture was completed to the OD600When the concentration is 75-85 ℃, adding IPTG (isopropyl-beta-D-thiogalactoside) for induction;
the inclusion body obtaining step described in step S2 is as follows: carrying out solid-liquid separation on the fermentation liquor to obtain thalli; resuspending the thalli in a crushing liquid, homogenizing and crushing the thalli in a water bath, centrifugally collecting precipitates, dissolving the precipitates in a washing buffer solution for washing, and centrifugally collecting the inclusion body precipitates;
the protease used in the enzyme digestion in the step S3 is one or at least two of bovine trypsin, porcine trypsin and 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 urea solution, diluting and then carrying out enzyme digestion by using protease.
14. The use of the method of claim 12 or 13 for producing the whitening polypeptide PKEK using genetic engineering techniques for the industrial production of the whitening polypeptide PEKE.
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