CN113201074B - PKEK fusion protein and preparation method and application thereof - Google Patents

PKEK fusion protein and preparation method and application thereof Download PDF

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CN113201074B
CN113201074B CN202110494341.9A CN202110494341A CN113201074B CN 113201074 B CN113201074 B CN 113201074B CN 202110494341 A CN202110494341 A CN 202110494341A CN 113201074 B CN113201074 B CN 113201074B
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CN113201074A (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 PKEK fusion protein has the following structural formula: (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 simultaneously, a plurality of whitening polypeptides PKEK and flexible connecting peptides are sequentially connected in series to form the fusion protein, so that misplaced impurities generated in the enzyme digestion process can be effectively reduced, further, 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 PKEK fusion protein inclusion bodies in a large amount within 12 hours, the expression quantity can reach 45g/L at most, and the preparation method is simple and efficient in process, easy to amplify and beneficial to industrial production.

Description

PKEK fusion protein and preparation method and application thereof
Technical Field
The invention belongs to the field of genetic engineering, and in particular relates to a PKEK fusion protein, a preparation method and application thereof.
Background
In vitro cell experiments prove that under the irradiation of ultraviolet UVB, the PKEK can effectively reduce the expression of interleukin-6, interleukin-8 and TNF-a and obviously reduce the expression of a pigmentation modulator (alpha-MSH). In another group of random double blind skin efficacy test, after 10 healthy subjects continuously use PKEK once a day for four weeks, the expression of the skin interleukin-1 alpha, interleukin-6, interleukin-8, tumor necrosis factor-alpha and tyrosinase of the PKEK tested group is obviously reduced under the irradiation of ultraviolet UVB compared with the control group; after the PKEK and the vitamin C sodium phosphate are combined for 6 weeks by 39 women in the Gaojia region, the facial spots are obviously faded, and the spot fading of the PKEK or the vitamin C sodium phosphate is not obvious when the PKEK or the vitamin C sodium phosphate is singly used; thus, both in vivo and in vitro experiments described above demonstrate that PKEK is effective in reducing facial pigmentation due to outdoor UV light.
At present, PKEK is mainly produced by adopting a liquid phase synthesis or solid phase synthesis (SPPS) method, and as the side chains of lysine and glutamic acid which are raw materials used in chemical synthesis are protected, a uncapping process is required after the peptide transfer reaction is finished, crystallization is performed again, the operation is complicated, racemization chiral impurities are easy to form in the chemical synthesis process, and the preparation cost of the high-purity PKEK is high. Therefore, the production of polypeptides by genetic engineering techniques has become a major trend.
Patent application CN107881187a discloses that the use of EEEEK, EDK, DDDDK as a linking peptide, through the formation of a salt bond between the side chain carboxyl of D or E and the side chain amino of K, reduces the acylation modification of the K site, however, introducing the linking peptide formed by D or E in front of K does not affect the cleavage efficiency of protease, and does not solve the problem of miscut of PKEK in the subsequent cleavage process.
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 the target protein expressed by inclusion bodies is denatured and renatured firstly and then subjected to tag technology purification; purifying the fusion protein by an ELPs label purification technology, adopting SUMO protease for cleavage, adding salt and centrifuging to remove the diad fusion label ELPs-SUMO, obtaining recombinant active peptide, and then adopting pepsin, trypsin and chymotrypsin for enzymolysis of the recombinant active peptide to ultrafiltrate the small peptide mixture. Although the method can prepare small peptide, the steps and the process are complicated, expensive SUMO protease with low enzyme digestion efficiency is needed, and denaturation and renaturation are needed for inclusion bodies before enzyme digestion. In addition, since SUMO protease is not tolerant to denaturing fluids, it is necessary to remove denaturing renaturation fluids using chromatographic techniques prior to cleavage, resulting in the cost of small peptides prepared by biosynthetic methods being not significantly advantageous over chemical synthesis methods.
Patent application CN106560475a discloses a genetic engineering biosynthesis process of an active short peptide, which comprises the steps of connecting the active short peptide in series, expressing the active short peptide with a purification tag in a microbial cell as a soluble protein or inclusion body protein, and degrading the recombinant protein by using high-specificity protease to finally form a single active short peptide. Although the method has certain advantages in the aspect of preparing single active short peptide, because PKEK contains a plurality of basic amino acids, the isoelectric point is higher after the PKEK is connected in series, for example, the theoretical isoelectric point of 10 PKEK polypeptides connected in series is 9.92, the polypeptide containing the basic amino acids is usually expressed in a form of soluble protein in microbial cells, inclusion body proteins are extremely difficult to form, so that the polypeptide is easy to degrade, lysine side chain amino in free PKEK polypeptides is easy to be acetylated and modified, and the method still cannot solve the problem of misplacement of PKEK polypeptides connected in series 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 primary aim of the invention is to overcome the defects and shortcomings of the prior art, and provide a PKEK fusion protein, wherein the fusion protein is expressed in a microbial cell in the form of inclusion body protein, and meanwhile, the fusion protein is formed by sequentially connecting a whitening polypeptide PKEK and a flexible connecting peptide in series, so that misplaced impurities generated in the enzyme digestion process can be effectively reduced, further, the high-purity whitening polypeptide PKEK can be efficiently obtained, and the industrial production is facilitated.
Another objective of the present invention is to provide a gene encoding the PKEK fusion protein.
It is still another object of the present invention to provide the use of the gene encoding the PKEK fusion protein described above.
The aim of the invention is achieved by the following technical scheme:
a PKEK fusion protein, which comprises a protein formed by sequentially connecting a plurality of whitening polypeptides PKEK and flexible connecting peptides in series, and has the following structural formula: (PKEK-B) n; wherein: b is flexible connecting peptide; n is an integer of 1 to 30.
The PKEK fusion protein has the following structural formula: A-D d - (PKEK-B) n; wherein: a is chaperonin; d is a protease cleavage site; b is flexible connecting peptide; n is an integer of 1 to 30; d is an integer of 0 to 10.
The n is preferably 10 to 30; more preferably 10 to 20.
D is preferably 1 to 5; further preferably 1.
The chaperonin is one or more of KSI, pagP, paP3.30, TAF12, GDF15, GYGB, mKSI, mPagP, mPA 3.30, mGDF15, mGYGB and mTAF12, and m represents a mutant. The function of the chaperone protein is to assist the fusion protein to form inclusion bodies in escherichia coli, promote the formation of the inclusion bodies, improve the yield and quality of the inclusion bodies and reduce the risks of degradation and side chain modification; and is convenient for 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 have high hydrophobicity, are easy to form inclusion bodies in escherichia coli, and can guide small peptide connected at the C end of the inclusion bodies to carry out high-efficiency expression, so that KSI, pagP, paP3.30, GDF15, GYGB or TAF12 proteins are selected as chaperones; in addition, since the wild-type chaperone protein has a plurality of lysine residues and arginine residues, too many wrong enzyme fragments are formed in the subsequent enzyme digestion process, if lysine and arginine in the wild-type chaperone protein are mutated into other amino acids, the formation of impurities can be effectively reduced, and the enzyme digestion purity can be remarkably improved, so the chaperone protein can also be a mutant of KSI, pagP, paP3.30, GDF15, GYGB or TAF12 protein, namely mKSI, mPAGP, mPA 3.30, mGDF15, mGYGB or mTAF12. The chaperone protein is a single chaperone or a combination of chaperones, 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 as shown in X m ---X 2 -X 1 The peptide sequences shown; wherein X is 1 X is protease cleavage site 2 To X m Is a flexible segment; m is an integer of 0 to 10, m is 0 representing the deletion of the flexible connecting peptide, m is 1, namely the flexible connecting peptide is only 1 amino acid, and so on; preferably, when m is 0, d cannot be 0, and n is 1 to 30; or m is 0 and d is 0, said n cannot be 1.
The protease cleavage site is Arg or Lys; preferably Lys.
The amino acid in the flexible fragment cannot be Pro, thr or Trp; preferably Gly, ala or Ser.
Preferably, m is an integer of 1 to 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 formed by fusing TAF12 with other chaperonins, 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; n is preferably 10 to 30; more preferably 10 to 20.
The other chaperone protein is preferably Pagp, KSI or GYGB.
When the chaperonin is KSI or formed by fusing KSI with other chaperonins, the flexible connecting peptide is preferably AK, GK, AAK, AAGK, AAGGK or GGAAK; more preferably GGAAK or GGSASK; the n is preferably 10 to 20.
The other chaperone protein is preferably Pagp or TAF12.
When the chaperonin is PagP or formed by fusing PagP with other chaperonins, the flexible connecting peptide is preferably AK, GK, AAK, AAGK, AAGGK, GGAAK or GGSASK; more preferably AAK; the n is preferably 10 to 20.
The other chaperone protein is preferably KSI or TAF12.
A gene encoding the above PKEK fusion protein, which can be obtained by codon usage; more preferably, the sequence is designed according to the codon usage preference of the host bacterium, 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 the sequence shown as 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 above-described gene encoding a PKEK fusion protein; the gene for encoding PKEK fusion protein is cloned into an expression vector.
The promoter on the expression vector comprises a T7 promoter, a T7Lac promoter, a Tac promoter, a Lac promoter and a Trp promoter; preferably the T7 promoter and the T7lac promoter.
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 preferred is pET-31b (+) or pET29a (+).
A recombinant expression strain comprising the recombinant vector; the recombinant vector is obtained by transferring the recombinant vector into a genetic engineering strain.
The genetically engineered strain is selected from bacteria, yeast and fungi; bacteria are preferred; more preferred is E.coli (Escherichia coli).
The PKEK fusion protein can be obtained by a chemical synthesis method or expressed by a genetic engineering method; preferably expressed by genetic engineering methods, the preparation method preferably comprises the following steps: fermenting and culturing the recombinant expression strain, and collecting inclusion bodies of fusion proteins to obtain PKEK fusion proteins.
The application of the PKEK fusion protein in preparing the whitening polypeptide PEKE comprises the following preferred specific steps: and dissolving and enzyme cutting inclusion bodies of the fusion protein, and separating and purifying to obtain the active whitening polypeptide PKEK.
A preparation method for producing a whitening polypeptide PEKE by using a genetic engineering technology comprises the following steps:
s1, obtaining a recombinant vector containing the PKEK fusion protein coding gene, wherein the structure of the coded fusion protein is as A-D d - (PKEK-B) n; wherein: a is chaperonin; b is a flexible connecting peptide, and the flexible connecting peptide is X m ---X 2 -X 1 The peptide sequence shown, X 1 X is protease cleavage site 2 To X m Is a flexible segment, m is an integer of 0 to 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 recombinant expression strain, and collecting inclusion body of fusion protein;
s3, dissolving and enzyme cutting inclusion bodies 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 bacteria are preferably E.coli. The PKEK fusion protein can be expressed in the form of inclusion bodies in escherichia coli, and the fusion protein does not need renaturation, and small molecular protein or polypeptide can be obtained by direct dilution and enzyme digestion after being dissolved under proper conditions, so that the technology is simple, the amplification is easy, and the method is suitable for industrial production.
The fermentation medium used in the fermentation described in step S2 consists of a nitrogen source, a carbon source and an inorganic salt.
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 to 3.5g/L of yeast powder, 4 to 6g/L, naCl of peptone and 0.5 to 1.5g/L, KH of peptone 2 PO 4 2.5～3.5g/L、Na 2 HPO 4 3～3.5g/L、MgSO 4 0.5～1.5g/L、(NH 4 ) 2 SO 4 5～7g/L、CaCl 2 ·2H 2 0.01-0.015 g/L of O, 4-4.2 g/L of glycerol, 0.8-1 mL/L of trace elements, and water as solvent; after sterilization, ammonia water is used for adjusting the pH value to 6.8-7.2; more preferably as follows: yeast powder 3g/L, peptone 5g/L, naCl g/L, KH 2 PO 4 3g/L、Na 2 HPO 4 3.25g/L、MgSO 4 1g/L、(NH 4 ) 2 SO 4 6g/L、CaCl 2 ·2H 2 0.01375g/L of O, 4.125g/L of glycerol and 0.875mL/L of trace elements, and the solvent is water; after sterilization, the pH was adjusted to 7.0 with ammonia.
The trace element mixed solution (total volume 100 mL) comprises the following components: feCl 2 ·4H 2 O 2～2.5g、ZnCl 2 0.1～0.15g、 CoCl 2 ·6H 2 O 0.15～0.25g、Na 2 Mo 4 ·2H 2 O 0.15～0.25g、CaCl 2 ·2H 2 O 0.05～0.15g、CuCl 2 ·2H 2 O 0.1～ 0.15g、H 3 BO 4 0.03～0.06g、MnSO 4 ·H 2 0.2-0.23 g of O, 9.5-10.5 mL of 37wt% concentrated hydrochloric acid, and water as solvent; the following are preferred: feCl 2 ·4H 2 O 2.287g、ZnCl 2 0.131g、CoCl 2 ·6H 2 O 0.2g、Na 2 Mo 4 ·2H 2 O 0.2g、CaCl 2 ·2H 2 O 0.1g、CuCl 2 ·2H 2 O 0.125g、H 3 BO 4 0.05g、MnSO 4 ·H 2 O0.217 g, 37wt% concentrated hydrochloric acid 10mL, the solvent was water.
The fermentation mode is high-density fermentation.
The specific steps of the fermentation are preferably as follows: fermenting at 36-38 deg.c and pH 6.8-7.2 with Dissolved Oxygen (DO) 5-50% maximum oxygen saturation; feeding is started when the culture is carried out until the carbon source is exhausted, and the culture is carried out until the concentration of the bacterial cells OD 600 75-85, adding IPTG for induction.
The feed medium used for the feed comprises the following components: 450-550 g/L glycerol, 30-40 g/L yeast extract powder, 60-70 g/L yeast peptone, 3-4 mL/L trace elements and the balance of water; the preferred composition is as follows: 500g/L glycerol, 33g/L yeast extract powder, 66/L g yeast peptone, 3.5mL/L trace elements and the balance of water.
The feeding rate is preferably 0.4 mL-min -1 ·L -1 The method comprises the steps of carrying out a first treatment on the surface of the More preferably 0.3 to 0.5 mL/min -1 ·L -1 。
The OD 600 80.
The final concentration of IPTG in the fermentation system is 0.2-0.4 mM; more preferably 0.3mM.
The induction time is at least 3 hours; more preferably 12 to 15 hours.
The inclusion body in the step S2 is obtained by crushing the bacterial cells by a high-pressure homogenization method.
The inclusion body obtaining step in step S2 is preferably as follows: performing solid-liquid separation on the fermentation broth to obtain thalli; and (3) suspending the bacterial body in a crushing liquid, homogenizing the crushed bacterial body under a water bath, centrifuging to collect precipitate, dissolving the precipitate in a washing buffer solution for washing, and centrifuging to collect inclusion body precipitate.
The solid-liquid separation is preferably performed by centrifugation.
The thallus and the crushing liquid are preferably prepared according to the weight ratio of 1g: 5-10 mL of the mixture is mixed; more preferably at 1g:7mL of the mixture is mixed.
The composition of the crushing liquid is preferably as follows: 6 to 6.2g/L Tris and 1.4 to 1.5g/L EDTA, and the balance being purified water, and adjusting the pH value to 7.8 to 8.2; more preferably as follows: 6.05g/L Tris and 1.46g/L EDTA, balance purified water, pH 8.0.
The conditions for the homogenization are preferably 900bar.
The precipitation and the washing buffer are preferably present in an amount of 1g: mixing 10-20 mL; more preferably at 1g:15mL of the mixture is mixed.
The composition of the wash buffer is preferably as follows: 6 to 6.1g/L Tris, 1.4 to 1.5g/L EDTA, 2.8 to 3.0g/L NaCl, 9 to 11mL/L TritionX-100 and the balance of water; the following are preferred: 6.05g/L Tris, 1.46g/L EDTA, 2.9g/L NaCl, 10mL/L TritionX-100, the balance being water.
The protease used in the cleavage 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 inclusion bodies of the fusion protein in the step S3 is as follows: the inclusion bodies of the collected fusion proteins are dissolved by urea solution, diluted and then subjected to enzyme digestion by protease.
The concentration of the urea solution is 5M or more, more preferably 8M.
The consumption of the urea solution is preferably calculated according to the proportion of 1g inclusion body to 5.5-6.5 mL urea solution; more preferably, the ratio is calculated by mixing 1g of inclusion body with 6mL of urea solution.
The preparation method for producing the whitening polypeptide PKEK by using 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 using 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:
the inventor sequentially connects PKEK and flexible connecting peptide in series, so that the cleavage efficiency of the second lysine in PKEK is obviously higher than that of the first lysine, a large number of PKEK complete polypeptide molecules are finally obtained, and misplaced impurities PK and EK are obviously reduced; meanwhile, due to the fact that the connecting peptide is introduced, partial misplaced impurities exist in the form of connecting peptide+PK and EK+connecting peptide, so that the misplaced impurities are obviously different from the physicochemical properties of the complete PKEK whitening polypeptide, the impurities can be effectively removed through a chromatographic process, and the misplaced impurities are controlled at a lower level.
The invention provides a PKEK fusion protein, which is expressed in a microbial cell in the form of inclusion body protein, and meanwhile, the fusion protein is formed by sequentially connecting a whitening polypeptide PKEK and a flexible connecting peptide in series, so that misplaced impurities generated in the enzyme digestion process can be effectively reduced, further, 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 PKEK fusion protein inclusion bodies in a large amount within 12 hours, the expression quantity can reach 45g/L at most, and the preparation method is simple and efficient in process, easy to amplify and beneficial to industrial production.
Drawings
FIG. 1 is a recombinant plasmid pET31b-TAF12-K- (PKEK-AAK) described in example 1 10 Plasmid map of (3).
FIG. 2 is a genetically engineered strain BL21 (DE)/pET 31b-TAF12-K- (PKEK-AAK) provided in example 1 10 Sample SDS-PAGE (SDS-PAGE) chart obtained by shaking fermentation induction for 3h; wherein lane M is a protein marker, lane 1 is a negative control, lanes 2-7 represent different monoclonal antibodies.
FIG. 3 is a genetically engineered strain BL21 (DE)/pET 31b-TAF12-K- (PKEK-AAK) provided in example 1 10 Sample SDS-PAGE (SDS-PAGE) chart obtained by shaking fermentation induction for 15 h; wherein lane M is a protein marker, lane 1 is a negative control, lanes 2-7 represent different monoclonal antibodies.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Material
Restriction enzymes NdeI and XhoI were purchased from NEB company; TAF12-K- (PKEK-AAK) 10 Fusion gene sequence (shown as SEQ ID NO. 4) is entrusted to synthesis by Nanjing Jinsri company; agarose gel recovery kits were purchased from Shanghai Co., ltd; DNA Ligation Kit was purchased from TaKaRa; coli DH 5. Alpha. Was purchased from Life Technologies; plasmid extraction kits were purchased from Shanghai Co., ltd; identification of the compositions containing TAF12-K- (PKEK-AAK) 10 Cloning of the gene fragment delegated sequencing by Invitrogen corporation; coli BL21 (DE 3) was purchased from Life technologies; the improved Lowry method protein concentration determination kit is purchased from Shanghai Co., ltd; the stirred tank fermentor was purchased from Oriental biology Inc.; recombinant bovine trypsin was purchased from Shanghai Yaxin Biotechnology Co., ltd; recombinant porcine trypsin was purchased from Roche's reagent; recombinant lysyl endonucleases were purchased from japan and light purity chemicals, inc; uni PMM30-500 filler was purchased from Soy micro technology Co., ltd.
Example 1
A preparation method for producing a whitening polypeptide PKEK by using a genetic engineering technology comprises the following steps:
1. construction of recombinant plasmid pET31b-TAF12-K- (PKEK-AAK) 10
1.1 Artificial Synthesis of a Gene sequence TAF12-K- (PKEK-AAK) containing chaperones, protease cleavage site, PKEK and connecting peptide 10
Designing gene sequence TAF12-K- (PKEK-AAK) containing molecular chaperones, protease cleavage site, PKEK and connecting peptide 10 According to codon usage preference of colibacillus, selecting codon with higher usage frequency, regulating GC content, removing cis-acting element and repeated sequence affecting gene transcription, at the same time introducing double-stop codon TAATGA at 3' end of gene sequence, in TAF12-K- (PKEK-AAK) for convenient gene operation 10 The NdeI enzyme cutting site sequence CATATG is introduced into the 5' end of the fusion gene sequence, and the fusion gene sequence is formed in the TAF12-K- (PKEK-AAK) 10 The 3' end of the fusion gene is introduced with XhoI restriction enzyme cutting site CTCGAG, and the optimized TAF12-K- (PKEK-AAK) 10 The sequence of the fusion gene is shown as SEQ ID NO. 4.
1.2pET31b-TAF12-K-(PKEK-AAK) 10 Construction of recombinant expression vectors
Plasmid pET-31b (+) was digested with restriction enzymes NdeI and XhoI, 5. Mu.L of the digested product was analyzed by agarose gel electrophoresis at a mass-to-volume ratio of 0.8%, after confirming that the digestion was completed, the whole digested product was subjected to agarose gel electrophoresis at a mass-to-volume ratio of 0.8%, a gel containing a DNA fragment of about 5.3kb was excised, and the DNA fragment in the gel was purified into 30. Mu.L of deionized water using an agarose gel recovery kit, thereby obtaining a plasmid fragment having a cohesive end. Similarly, the pair TAF12-K- (PKEK-AAK) was treated with NdeI and XhoI 10 Double enzyme digestion is carried out on the fusion gene fragment, and the DNA fragment after enzyme digestion is purified into 20 mu L of deionized water, so that the gene fragment with the sticky end is obtained. The gene fragment and the plasmid fragment were ligated using a DNA Ligation Kit (DNA Ligation Kit) at 16℃overnight, 10. Mu.L of the overnight ligated product was taken and added to 80. Mu.L of the Ligation Kit using CaCl 2 To E.coli DH 5. Alpha. Competent cells prepared by the method (third edition of molecular cloning laboratory Manual published by Cold spring harbor laboratory, U.S.A.), treated at 42℃for 90 seconds, 300. Mu.L of LB liquid medium (wherein, peptone 10g/L, yeast powder 5g/L, sodium chloride 5g/L, pH7.0 to 7.5) preheated at 37℃was rapidly added, and the culture was shake-cultured at a low speed (100 to 150 rpm) in a shaking table at 37℃for 45 minutes, and then 100. Mu.L of the culture was coated with LB solid medium (wherein, peptone 10g/L, yeast powder 5g/L, sodium chloride 5g/L, agar powder 15g/L, pH7.0 to 7.5) to which ampicillin (final concentration of 100. Mu.g/ml) was added. Culturing in an incubator at 37 ℃ for 18 hours in an inversion way until single colonies grow out, randomly picking part of the single colonies to carry out bacterial liquid PCR identification, wherein the PCR reaction conditions are as follows: 94 ℃ for 5min; 30 cycles at 94℃for 30s, 55℃for 30s, 72℃for 30 s; 72 ℃ for 5min; the primer is as follows: t7 promoter primer and 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 by using plasmid extraction kit for the positive clone obtained by preliminary screening, and extracting plasmid according to instruction, and performing enzyme digestion identification by NdeI and XhoI to obtain clone of gene fragment of about 465bp and vector fragment of about 5.3kb, namely TAF12-K- (PKEK-AAK) 10 Cloning of the Gene fragment. Will be identified as containing TAF12-K- (PKEK-AAK) 10 Cloning of the gene fragment was sequenced. The single clone which has no base mutation and frame shift is inoculated to 50mL LB liquid medium added with ampicillin (final concentration is 100 mug/mL), and cultured for 18h at 37 ℃ and 250rpm, the obtained culture is used for extracting plasmid by a plasmid extraction kit, namely a recombinant expression vector named pET31b-TAF12-K- (PKEK-AAK) 10 As shown in fig. 1.
2、BL21(DE3)/pET31b-TAF12-K-(PKEK-AAK) 10 Screening of expression strains and product identification
Competent cells of E.coli BL21 (DE 3) were prepared according to the calcium chloride method provided in the third edition of molecular cloning laboratory Manual published in Cold spring harbor laboratory. 1 μl of the recombinant expression vector pET31b-TAF12-K- (PKEK-AAK) of example 1 was used 10 The competent cells of E.coli described above were transformed by the same procedure as in 1.2 for calcium chloride. The transformation liquid is respectively coated on LB solid medium added with ampicillin (final concentration is 100 mu g/mL), and the transformation liquid is inversely cultured at 37 ℃ until single colony appears, thus obtaining PKEK fusion protein expression strain library, which is named BL21 (DE 3)/pET 31b-TAF12-K- (PKEK-AAK) 10 . Meanwhile, the competent cells of the escherichia coli BL21 (DE 3) are transformed by pET-31b (+) to obtain BL21 (DE 3)/pET 31b bacteria which serve as a negative control.
Picking single colony 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, shake culturing at 37deg.C and 250rpm, and obtaining bacterial liquid OD 600 When the bacterial strain is=0.5 to 1.0, 3 to 6mL of bacterial strain is inoculated to 100mL of LB liquid medium, 3 to 6mL of BL21 (DE 3)/pET 31b bacterial strain is inoculated to 100mL of LB liquid medium to serve as negative control, and the temperature is 37 DEG CShaking culture at 250rpm to OD 600 When the bacterial strain is=0.6-0.8, IPTG (isopropyl-beta-D-thiopyran galactoside with the final concentration of 1 mmol/L) is added for induction, bacterial solutions (4 mL) for 3h and 15h are respectively taken for induction, centrifugation is carried out at 12000rpm for 1 min, the supernatant is removed, and the bacterial strain is stored at the temperature of minus 20 ℃ for standby.
The frozen cells were removed at-20℃and resuspended in 5mL of urea solution at a concentration of 8mol/L, and sonicated in an ice-water mixture for 10 minutes (3 seconds and 5 seconds of sonication, and circulated as such). 15 mu L of the broken liquid is added into 15 mu L of the sample buffer solution, after fully mixing, 10 mu L of SDS-PAGE (wherein the concentrated Gel contains 5% of acrylamide-methylene bisacrylamide (prepared according to the volume ratio of 29:1) and the separated Gel contains 15% of acrylamide-methylene bisacrylamide by volume ratio of 29:1) is carried out, the electrophoresis condition is that the concentrated Gel is set to be 11mA, the separated Gel is set to be 22mA, after the electrophoresis is finished, the Gel is taken out, coomassie blue staining solution (containing 0.6g coomassie blue R-250, 450mL of ethanol and 100mL of glacial acetic acid per liter, and the balance of purified water) is used for staining overnight, the decolorized solution (containing 250mL of ethanol and 80mL of glacial acetic acid per liter and the balance of purified water) is used for decolorization until the background is transparent, then the Gel is photographed under a transparent background to extract an image, as shown in fig. 2 and 3, biological image processing software Gel-Pro Analyzer 4.0 is used for scanning and analyzing the proportion of a target band to the total band, namely the total protein of PK fusion protein in a total protein concentration of the total protein is measured by using a Lowry protein expression method, and the total protein concentration of the total protein is measured in a PKwry protein concentration of the total protein is measured by a total protein concentration of the total protein of the fusion protein in a dilution protein, and the total protein concentration of the total protein is measured by a total protein concentration of the total protein is measured.
According to the electrophoresis result, the genetically engineered bacterium BL21 (DE 3)/pET 31b-TAF12-K- (PKEK-AAK) 10 The target protein is not expressed before induction, which can effectively reduce the toxicity of the target protein to host cells and is beneficial to prolonging the expression time of the protein. The control bacterium BL21 (DE 3)/pET-31 b (+) without PKEK gene can express aldosterone isomerase with molecular weight of about 14kDa after IPTG induction, while the genetically engineered bacterium BL21 (DE 3)/pET 31b-TAF12-K-(PKEK-AAK) 10 A protein of approximately 17kDa can be expressed after IPTG induction, which corresponds to the theoretical molecular weight 16.947kDa of the PKEK fusion protein (obtained using the on-line software http:// web. Expasy. Org/computer_pi/prediction), indicating that the PKEK gene has been successfully introduced into genetically engineered bacteria.
The genetically engineered bacterium BL21 (DE 3)/pET 31b-TAF12-K- (PKEK-AAK) 10 Streaking on fresh LB solid medium containing ampicillin with final concentration of 100 μg/mL for pure culture, picking single colony, and culturing with LB liquid medium to OD 600 The bacterial liquid containing 15 percent of glycerol by volume percent is prepared by the method of (1.0-0), and the bacterial liquid is stored in a refrigerator at the temperature of-70 ℃ for standby.
3. PKEK fusion protein genetically engineered bacterium BL21 (DE 3)/pET 31b-TAF12-K- (PKEK-AAK) 10 Fermentation test
3.1 preparation of seed cultures
Taking 20 μL of BL21 (DE 3)/pET 31b-TAF12-K- (PKEK-AAK) cryopreserved at-70 ℃ 10 Inoculating strain to 50mL of LB liquid medium added with ampicillin (final concentration is 100 μg/mL), culturing in shaking table at 28deg.C and 250rpm for 16 hr to activate strain, inoculating 50mL of activated strain to 400mL of LB liquid medium, continuously culturing at 28deg.C and 250rpm for 3 hr to obtain seed culture, and controlling thallus concentration OD 600 Between 2.0 and 3.0.
3.2 Fermentation culture in 20L fermentor
Using a 20L stirred tank fermenter, the fermentation medium formulation shown in table 1 was sterilized and then fed with ammonia to adjust the pH to 7.0, the feed volume was 8L, and the fermentation conditions were controlled strictly: the temperature is controlled to 37 ℃, the pH is controlled to 7.0, the Dissolved Oxygen (DO) is controlled to be between 5 and 50 percent of the maximum oxygen saturation (the correction condition of the oxygen saturation is saturated sodium sulfite solution, 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 and 700rpm, the specific fermentation rotating speed is regulated and controlled according to the change of DO, the air flow rate is controlled to be between 4 and 20L/min, and the feeding is started when the carbon source is exhausted during the culture, wherein each kilogram of feed liquid comprises the following components: 500g glycerin, 33g yeast extract powder, 66g yeast eggWhite peptone, 3.5mL trace elements and the balance of water, wherein the whole process is 0.4mL min -1 ·L -1 And (3) feeding at a constant speed. When cultured to the concentration OD of the bacterial cells 600 Approximately equal to 80, IPTG addition was started to a final concentration of 0.3mM and induction was started.
Table 1: fermentation medium formula
Component (A) Dosage (g)
Yeast powder (Yeast Extract) 24.0
Peptone (Tryptone) 40.0
NaCl 8.0
KH 2 PO 4 24.0
Na 2 HPO 4 26.0
MgSO 4 8.0
(NH 4 ) 2 SO 4 48.0
CaCl 2 ·2H 2 O 0.11
Glycerol 33.0
Microelement mixture (see Table 2) 7mL
Table 2: recipe of trace element mixed liquid (100 mL)
Component (A) Dosage (g)
FeCl 2 ·4H 2 O 2.287
ZnCl 2 0.131
CoCl 2 ·6H 2 O 0.2
Na 2 Mo 4 ·2H 2 O 0.2
CaCl 2 ·2H 2 O 0.1
CuCl 2 ·2H 2 O 0.125
H 3 BO 4 0.05
MnSO 4 ·H 2 O 0.217
37wt% concentrated hydrochloric acid 10mL
After induction for 12h and 1mL of fermentation broth was taken for 0h and 12h, respectively, and 10-fold dilution was performed with purified water, TAF12-K- (PKEK-AAK) was measured 10 Expression level of fusion protein (two parallel batches). Genetically engineered strain
BL21(DE)/pET31b-TAF12-K-(PKEK-AAK) 10 The average yield of inclusion bodies containing the fusion protein after conversion in the fermentation test was 45g/L (obtained by the inclusion body measurement obtained by the separation in the following step 4.1), wherein the content of the fusion protein in the inclusion bodies was 44%, i.e., 19.8g/L.
4. Extraction and cleavage of PKEK fusion protein inclusion body
4.1 isolation and extraction of inclusion bodies
Collecting the bacterial cells from the fermentation broth obtained in the step 3 by centrifugation at 4500rpm for 30 minutes, suspending the bacterial cells in a crushing solution (containing 6.05g Tris and 1.46g EDTA per liter, the balance being purified water, and adjusting pH to 8.0) at a mass/volume ratio of 1:7, homogenizing the crushed bacterial cells under high pressure (900 bar) in a water bath, collecting the precipitate by centrifugation, dissolving the collected precipitate in a washing buffer (containing 6.05g Tris, 1.46g EDTA, 2.9g NaCl, 10mL TriionX-100 per liter, and the balance being purified water) at a mass/volume ratio of 1:15, washing, and centrifugally collecting the inclusion body precipitate.
4.2 enzyme digestion of inclusion body denaturation solution
After 2.5g of the above-collected inclusion body was dissolved in 15mL of a denatured solution (containing 8mol of urea per liter and the balance purified water), the solution was diluted 1-fold with 5mM Tris, and the pH was adjusted to 9.0, followed by stirring at room temperature for 2 to 5 hours. And (3) adopting recombinant bovine trypsin to carry out enzyme digestion according to the instruction, and obtaining a PKEK crude product through enzyme digestion.
Liquid phase detection conditions for PKEK crude product: BEH-C8 (packing size 1.7 μm particle size, column size 2.1X100 mm) analysis column, column temperature 35℃and flow rate 0.2 mL/min. Mobile phase a: an aqueous solution containing 0.1% (v/v) TFA; mobile phase B: acetonitrile containing 0.09% (v/v) TFA; gradient: 0-25 min, and increasing the phase B from 30% to 80%; 25-38 min: phase B increased from 80% to 100%. The PKEK peak-exiting time is 21.5min, the statistical peak area occupied ratio is 13.2%, namely the purity of the PKEK crude product is 13.2%.
5. Purification of the cleaved PKEK crude product
100mL of the digested PKEK crude product prepared in step 4 above was loaded onto a chromatography column packed with 10mL of Uni PMM30-500 (which was previously equilibrated with a buffer solution containing 0.1% (v/v) TFA in 20% isopropanol) and after loading was completed, rinsed to baseline plateau with equilibration buffer (buffer solution containing 0.1% (v/v) TFA in 20% isopropanol). Then, the solution is eluted with a linear gradient of 100mL of isopropanol A with the concentration of 20% (v/v) (containing 0.1% (v/v) TFA buffer) and 100mL of isopropanol B with the concentration of 40% (v/v) (containing 0.1% (v/v) TFA buffer), and the elution flow rate is less than or equal to 2 times of column volume per hour. And collecting main peaks in a segmented way, wherein about 20% of peak heights are collected, and the detection wavelength is 280nm, so that PKEK is obtained. The purity of PKEK prepared by the purification process reaches 95.4 percent. PKEK liquid phase detection conditions: BEH-C8 (packing size 1.7 μm particle size, column size 2.1X100 mm) analysis column, column temperature 35℃and flow rate 0.2 mL/min. Mobile phase a: an aqueous solution containing 0.1% (v/v) TFA; mobile phase B: acetonitrile containing 0.09% (v/v) TFA; gradient: 0-25 min, and increasing the phase B from 30% to 80%; 25-38 min: phase B increased from 80% to 100%. Mass spectrometry identification: the peak of PEKE is taken by a Simer high-resolution liquid chromatography mass spectrometer for high-resolution mass spectrometry analysis, and deconvolution is carried out by using a Thermo BiopharmaFinder2.0, and the acquisition range is as follows: MS 100-1000. The result shows that the molecular weight of PKEK prepared by the method 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 basically the same as that of example 1, except that the protease used in the cleavage of this example is recombinant porcine trypsin, the reaction is carried out for 2 hours at 16 ℃ and then the acid regulation and termination reaction are carried out, and the PKEK crude product is obtained after the cleavage, and the purity of the PKEK crude product is 13.2%.
Example 3
The method of this example is basically the same as that of example 1, except that the protease used in the cleavage of this example is recombinant lysyl endonuclease, and the cleavage reaction is carried out for 2 hours at 25 ℃ and then the acid regulation is terminated, and the crude PKEK product is obtained by cleavage, and the purity of the crude PKEK product is 13.5%.
Example 4
This example is essentially the same as the method described in example 1, except that the recombinant expression vector selected in this example has a different number of chaperones, connecting peptides and PKEK in tandem. The relative yields of the different chaperones and connecting peptide fusion proteins are shown in Table 3, and the relative purities of PKEK after cleavage are shown in Table 4.
Table 3: fermentation yield test of different molecular chaperones and connecting peptide fusion proteins
Figure BDA0003053671650000071
Description: the relative yields in the above table refer only to the relative yields of the PKEK portion of the fusion protein molecule, calculated with the subtraction of chaperones, protease cleavage sites and linker peptides.
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 in SEQ ID NO. 29; the amino acid sequence of PagP molecular chaperone is shown in SEQ ID NO. 24; the amino acid sequence of the TAF12 molecular chaperone is shown in SEQ ID NO. 1; the amino acid sequence of 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 as 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 nucleic acid sequences is an enzyme cutting site NdeI, the 3' -end is a stop codon (taatga) and an enzyme cutting site XhoI, the first amino acid of the initial amino acid sequence is M, and the first amino acid is from the NdeI enzyme cutting site. The results in Table 3 demonstrate that the yield of PKEK increases significantly after addition of chaperones. Co-expression of certain hydrophobic chaperones may further increase PKEK production.
The relative purities of PKEK monomers obtained by cleavage of the different inclusion bodies were shown in Table 4 as in example 1.
Table 4: relative purity of PKEK after cleavage of fusion proteins of different chaperones and connecting peptide
Molecular chaperones Protease cleavage site Connecting peptide PKEK-connecting peptide tandem number PKEK monomer relative purity
TAF12 K Without any means for 10 38％
TAF12 K Without any means for 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: the PKEK content in inclusion bodies was the same at all groups of the above table, except for chaperones and connecting peptides. The percentage indicates the relative purity after cleavage, and the higher the purity, the smaller the proportion of miscut.
The results in table 4 illustrate: in the absence of the linker peptide, cleavage yields a monomeric PKEK that is much lower than the molecule with the linker peptide. At the same time, when X in the peptide is linked m ---X 2 When the polypeptide is A, G, AA, AAG, GGAA, GGSAS and other small side chain hydrophobic flexible polypeptides, PKEK monomers obtained after the cleavage are far higher than WT, PP and other large side chain hydrophobic polypeptides. The large side chain hydrophobic polypeptides such as WT, PP and the like are connecting peptides, and the yield of PKEK monomers after enzyme digestion is only equivalent to that of a group without connecting peptides. Therefore, the connecting peptide provided by the invention can greatly improve the cleavage yield of PKEK monomers.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
<110> Pinghai 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 (8)

1. A PKEK fusion protein, characterized by: the PKEK fusion protein has the following structural formula: A-D d - (PKEK-B) n; wherein: a is chaperonin; d (D) d Is a protease cleavage site; b is flexible connecting peptide; n is an integer of 10 to 30; d is 1;
the flexible connecting peptide is AK, GK, AAK, AAGK, GGAAK or GGSASK;
k in the flexible connecting peptide is a protease cleavage site;
said D d Is K;
when the chaperonin is TAF12, the flexible connecting peptide is AK, GK, AAK, AAGK or GGAAK;
when the chaperonin is formed by fusing TAF12 and Pagp, the flexible connecting peptide is AAK;
when the chaperonin is formed by fusing TAF12 and KSI, the flexible connecting peptide is AAK;
when the chaperonin is formed by fusion of TAF12 and GYGB, the flexible connecting peptide is AAK;
when the chaperonin is KSI, the flexible connecting peptide is GGAAK or GGSASK;
when the chaperonin is PagP, the flexible connecting peptide is AAK;
when the chaperonin is formed by fusing KSI and PagP, the flexible connecting peptide is AAK;
when the chaperonin is Pap3.30, GDF15 or GYGB, the flexible connecting peptide is AAK.
2. A gene encoding the PKEK fusion protein of claim 1.
3. A recombinant vector, characterized in that: a gene comprising the gene of claim 2; is obtained by cloning the gene according to claim 2 into an expression vector.
4. A recombinant expression strain characterized in that: a recombinant vector according to claim 3.
5. Use of the PKEK fusion protein of claim 1 in the preparation of a whitening polypeptide PKEK.
6. A preparation method for producing a whitening polypeptide PKEK by using a genetic engineering technology is characterized by comprising the following steps:
s1, obtaining a recombinant vector comprising a gene encoding the PKEK fusion protein of claim 1;
s2, transferring the recombinant vector obtained in the step S1 into a genetic engineering strain to obtain a recombinant expression strain; fermenting and culturing recombinant expression strain, and collecting inclusion body of fusion protein;
s3, dissolving and enzyme cutting inclusion bodies of the fusion protein, and separating and purifying to obtain the active whitening polypeptide PKEK.
7. The method for preparing the whitening polypeptide PKEK by using genetic engineering technology according to claim 6, wherein the method comprises the following steps:
the genetically engineered strain in the step S2 is escherichia coli;
the composition of the fermentation medium used in the fermentation is as follows: 2.5-3.5 g/L yeast powder, 4-6 g peptone/L、NaCl 0.5～1.5g/L、KH 2 PO 4 2.5～3.5g/L、Na 2 HPO 4 3～3.5g/L、MgSO 4 0.5～1.5g/L、(NH 4 ) 2 SO 4 5～7g/L、CaCl 2 •2H 2 0.01-0.015 g/L of O, 4-4.2 g/L of glycerol, 0.8-1 mL/L of trace elements and water as solvent; after sterilization, ammonia water is used for adjusting the pH value to 6.8-7.2;
the trace elements comprise the following components: feCl per 100mL 2 •4H 2 O 2～2.5g、 ZnCl 2 0.1～0.15g、CoCl 2 •6H 2 O 0.15～0.25g、 Na 2 Mo 4 •2H 2 O 0.15～0.25g、CaCl 2 •2H 2 O 0.05～0.15g、 CuCl 2 •2H 2 O 0.1～0.15g、H 3 BO 4 0.03～0.06g、MnSO 4 •H 2 0.2 to 0.23g of O, 9.5 to 10.5 g of 37wt percent concentrated hydrochloric acid and mL, wherein the solvent is water;
the specific steps of the fermentation are as follows: fermenting at 36-38 deg.c and pH 6.8-7.2 and maximum oxygen saturation of 5-50% of dissolved oxygen; feeding is started when the culture is carried out until the carbon source is exhausted, and the culture is carried out until the concentration of the bacterial cells OD 600 Adding IPTG for induction when the amount is 75-85;
the inclusion bodies described in step S2 were obtained as follows: performing solid-liquid separation on the fermentation broth to obtain thalli; suspending the bacterial body in a crushing liquid, homogenizing the crushed bacterial body under water bath, centrifugally collecting sediment, dissolving the sediment in a washing buffer solution for washing, centrifugally collecting inclusion body sediment;
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 inclusion bodies of the fusion protein in the step S3 is as follows: the inclusion bodies of the collected fusion proteins are dissolved by urea solution, diluted and then subjected to enzyme digestion by protease.
8. The use of the method for producing a whitening polypeptide PKEK according to claim 6 or 7 by genetic engineering techniques for the industrial production of a whitening polypeptide PKEK.
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