CN100338091C - Encoding blended protein DNA and preparing of useful polypeptides by expression thereof - Google Patents
Encoding blended protein DNA and preparing of useful polypeptides by expression thereof Download PDFInfo
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- CN100338091C CN100338091C CNB991207483A CN99120748A CN100338091C CN 100338091 C CN100338091 C CN 100338091C CN B991207483 A CNB991207483 A CN B991207483A CN 99120748 A CN99120748 A CN 99120748A CN 100338091 C CN100338091 C CN 100338091C
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Abstract
The present invention relates to DNA for coding a nucleotide sequence of fusion protein, a carrier having the DNA, germs containing the carrier and a method for preparing useful polypeptide by cultivating the germs, wherein the germs belong to bacillus, and the fusion protein comprises a signal peptide sequence of bacillus cell wall protein, a mark sequence used for fusion protein separation and purification, a joint sequence, a sequence used for chemical or enzymatic schizolysis and an extraneous polypeptide sequence; the sequences are mutually and linearly connected in turn, the signal peptide sequence, the mark sequence and the joint sequence are optional sequences, and a nucleotide sequence is connected with the 3'-tail end of a nucleic sequence which comprises a bacillus promoter region.
Description
The present invention relates to the to encode DNA of new fusion rotein and these DNA are used for producing the purposes of the biologically active polypeptides that can utilize in pharmacy, scientific research and other industries.
Peptide material for example as the hormone of medicine and biologically active substance and be used to diagnose, the enzyme of industrial application and scientific research often obtains from organism by extraction method.Yet it but is difficult obtaining a large amount of pure materials by extraction method with low cost.Recently, because the progress of gene recombination technology, can prepare highly purified recombinant protein more economically, in large quantities by using from the various cells of organism such as microorganism, animal and plant.
But, also do not realize the mass production of the economy of useful proteins (or polypeptide) up at present as yet fully, so the exploitation of new technology also continuously carried out always.In addition, the present mass production system of developing can not prepare the protein of all kinds by gene recombination technology, and therefore in fact they are developed respectively according to proteinic kind.
In the expression of recombinant proteins system that adopts bacillus brevis, when foreign protein is connected the downstream of microbial cell wall-held protein (hereinafter referred for " CWP ") signal peptide and during with the expressing fusion protein of gained, the foreign protein with natural structure downcuts (Japanese Patent No.2082727 from the CWP signal peptide of desiring to be secreted into the substratum; JP-A-62-201583; People such as Yamagata H., bacteriology magazine, 169:1239-1245 () 1987); Udaka J., Japanese bio-science, biotechnology and agrochemistry association magazine, 61:669-676 (1987); People such as Takano M., microbiology biotechnology applications, 30:75-80 (1989); With people such as YamagataH., Proc.Natl.Acad.Sci.USA 86:3589-3593 (1989)).When with human epidermal growth factor's (hereinafter referred is " EGF ") when expressing in above-mentioned expression system, the high 10-100 of amount that the expression amount of EGF is expressed in other expression systems than EGF doubly; Expressed albumen keeps the active of its script and is secreted in the substratum; Therefore, easily expressed albumen is separated and purifying; And different with some escherichia expression systems, this system does not need inactivating protein is transformed into the complicated process of activated protein.In view of these reasons, above-mentioned expression system attracts much attention as the mass production system of recombinant protein.
But not all albumen that links to each other with the CWP signal peptide all can suitable amount be expressed with expressing EGF, and they always can not get off on desiring to be secreted into the signal peptide the substratum in cracking.
People such as Miyauchi have advised result of the above problems at " summary is talked in the annual meeting of Japanese bio-science, biotechnology and agrochemistry association " 67:372 in (1993).Promptly, 17 amino acid (have 9 or 12 are amino acid whose not to be had successfully) that they have prepared a kind of MWP protein N terminal are inserted in the encoding gene of the fusion rotein between MWP signal peptide and the flatfish growth hormone protein, and this gene is expressed in genus bacillus to obtain fusion rotein.Yet the albumen that is produced is not the protein of natural type, its N-terminal more amino acid.People such as Miyauchi point out that this expression is subjected to influence from the amino acid no purpose of the N-terminal of MWP.
People such as Miyauchi had not both expressed does not have hint to produce the polypeptide that has the same amino acid composition with corresponding natural type albumen by introduce chemistry or enzymatic lysis site in sequence yet.In fact, such cracking is difficult, because flatfish tethelin comprises that some are easily by the sequence of chemistry or enzymatic lysis.
In this case, for industrial purposes, develop and a kind ofly allogenic polypeptide is easily expressed and the excretory technology is exceedingly useful in the genus bacillus expression system, promptly, the high expression level system that develops a kind of recombinant protein is exceedingly useful, and wherein polypeptide has the sequence same with the polypeptide of natural type.
The object of the present invention is to provide a kind of genus bacillus expression system that comprises the coding DNA of fusion rotein, described fusion rotein contains useful peptide sequence, this system has the ability of energy great expression and secretion fusion rotein, and optionally the described fusion rotein of cracking is to obtain having the polypeptide of natural type structure.
The invention provides a kind of DNA that comprises the nucleotide sequence of encoding fusion protein, wherein said fusion rotein comprises: the sequence of being made up of one or more amino-acid residues of cell wall protein (CWP) N-terminal of genus bacillus, sequence that is used for chemistry or enzymatic lysis and the allogenic polypeptide sequence be made up of one or more amino-acid residues; Described these sequences are linear according to the order of sequence each other to be connected, and wherein said nucleotide sequence links to each other with 3 ' of the nucleotide sequence that comprises the bacillus promoter district-end.
Term used herein " one or more amino-acid residues of (cell wall protein) N-terminal " is meant the sequence of being made up of the one or more amino acid that begin number from the 1st amino acid of N-terminal.For example, the sequence of being made up of 3 amino acid is meant the 1st to the 3rd aminoacid sequence that amino acid is formed by cell wall protein.
Fusion rotein also can comprise the genus bacillus CWP signal peptide sequence that is positioned at N-terminal.
Fusion rotein also can comprise sequence of forming as the amino acid of separation and purifying mark and/or the aminoacid sequence that is used as joint.
In one embodiment of the invention, bacillus is a bacillus brevis.
As the amino-acid residue that is used for chemical cracking, for example be methionine(Met).In this case, fusion rotein should not contain other methionine residues again, so that obtain the highest specificity in chemical cracking reaction (the chemical cracking reaction of for example carrying out with cyanogen bromide).
The amino-acid residue that is used for enzymatic lysis can comprise can be by albumen enzymatic cracked sequence.Described examples of proteases is TEV proteolytic enzyme, V8 proteolytic enzyme or the like.
In first preferred embodiment of the present invention, fusion rotein comprises: the sequence of forming by the one or more amino-acid residues that are derived from the MWP protein N terminal, by 6 histidine residues form as separate and the sequence of the mark of purifying, as the aminoacid sequence Gly SerPro Val Pro Ser Gly of joint, be used for come out required methionine residues and do not contain the peptide sequence of methionine(Met) of desired polypeptides chemical cracking at its aminoacid sequence, wherein said MWP albumen is a kind of of cell wall protein; Described these sequences are linear according to the order of sequence each other to be connected.
In this case, fusion rotein can be included in the MWP signal peptide sequence of N-terminal.An example of described polypeptide is the proinsulin human.The sequence preference of being made up of one or more amino-acid residues of MWP protein N terminal comprises 6,7,8,9,10,11,12,13,14,15,17,20 or 50 amino acid.
In second embodiment preferred of the present invention, fusion rotein comprises: by 10 or 20 sequences that amino-acid residue is formed being derived from the MWP protein N terminal, by 6 histidine residues form as separating and the sequence of the mark of purifying, human epidermal growth factor's sequence as joint, come out the desired polypeptides cracking required aminoacid sequence Asp Tyr Asp Ile Pro Thr Thr Glu Asn Leu Tyr Phe Gln (SEQ ID NO:2) with TEV proteolytic enzyme, with in its aminoacid sequence, do not contain the TEV protease recognition sequence but its N-terminal have glycine or Serine peptide sequence, wherein said MWP albumen is a kind of of cell wall protein; Described these sequences are linear according to the order of sequence each other to be connected.
In the case, fusion rotein also can comprise the MWP signal peptide sequence that is positioned at N-terminal.As for polypeptide, what can give an example is human somatotropin's statin.
In the 3rd embodiment preferred of the present invention, fusion rotein comprises: the sequence of forming by 20 amino-acid residues that are derived from the MWP protein N terminal, by 6 histidine residues form as separate and the sequence of the mark of purifying, as the aminoacid sequence Gly SerPro Val Pro Ser Gly of joint, the desired polypeptides cracking is come out aminoacid sequence Phe Leu Glu required and the peptide sequence that does not contain L-glutamic acid in its aminoacid sequence with V8 proteolytic enzyme, wherein said MWP albumen is a kind of of cell wall protein; Described these sequences are linear according to the order of sequence each other to be connected.
In this case, fusion rotein also can comprise the MWP signal peptide sequence that is positioned at N-terminal similarly.Porcine glucagon is a kind of useful described polypeptide.
The invention still further relates to a kind of DNA that comprises the nucleotide sequence of encoding fusion protein, wherein said fusion rotein comprises: the CWP signal peptide sequence of genus bacillus, sequence that is used for enzymatic lysis and the allogenic polypeptide sequence be made up of amino-acid residue; Described these sequences are linear according to the order of sequence each other to be connected, and wherein said nucleotide sequence links to each other with 3 ' of the nucleotide sequence that comprises the bacillus promoter district-end.
In this invention, described signal peptide can directly link to each other with the sequence that the one or more amino acid from the CWP protein N terminal that are positioned at its downstream are formed.
Described bacillus is preferably bacillus brevis.
In one embodiment of the invention, comprise can be by albumen enzymatic cracked sequence for the sequence of being made up of the amino-acid residue that is used for enzymatic lysis.
In another embodiment of the invention, fusion rotein comprises: the signal peptide sequence of MWP, with TEV proteolytic enzyme the desired polypeptides cracking is come out aminoacid sequence Asp TyrAsp Ile Pro Thr Thr Glu Asn Leu Tyr Phe Gln required and the peptide sequence that does not contain the TEV protease recognition sequence in its aminoacid sequence, wherein said MWP albumen is a kind of of cell wall protein; Described these sequences are linear according to the order of sequence each other to be connected.
In the case, described signal peptide can directly link to each other with the sequence by forming from one or more amino acid of CWP protein N terminal that is positioned at its downstream.As described polypeptide, can enumerate the human growth hormone that has the sudden change of glycine or Serine at its N-terminal.
The present invention also provides the carrier that comprises above-mentioned a kind of DNA.
The present invention also further provides the bacterium that belongs to bacillus that is transformed by above-mentioned carrier.Preferred bacterium is a bacillus brevis.
The present invention also provides a kind of method for preparing recombinant polypeptide, and this method comprises: cultivate the bacterium of definition as mentioned in substratum, gather to make the fusion rotein that comprises allogenic polypeptide outside this bacterial cell; From substratum, isolate fusion rotein; From the isolated fusion rotein of institute, described allogenic polypeptide cracking is come out; With the described polypeptide of collection.
Present invention resides in as disclosed all or part content in the specification sheets of the Japanese patent application No.10-87339 of the application's priority document and/or the accompanying drawing.
Brief description of drawings
Fig. 1 shows fusion protein product MWPsp-MWPmp10-(His)
6The nucleotide sequence of the insulinogenic aminoacid sequence of-joint-Met-and this product of coding.
Fig. 2 shows the nucleotide sequence of the insulinogenic aminoacid sequence of fusion protein product MWPsp-MWPmp10-Met-and this product of coding.
Fig. 3 shows fusion protein product MWPsp-MWPmp20-(His)
6The nucleotide sequence of the aminoacid sequence of-EGF-TEV-somatostatin and this product of coding.
Fig. 4 shows fusion protein product MWPsp-MWPmp20-(His)
6The nucleotide sequence of the aminoacid sequence of-joint-V8-hyperglycemic-glycogenolytic factor and this product of coding.
Fig. 5 shows the synoptic diagram of each fusion dna being introduced the mode of bacillus brevis expression vector (pNU211R2L5).
Fig. 6 is the photo that shows the electrophoresis result that contains the insulinogenic substratum that links to each other with the His-mark that produces by the cultivation transformant, wherein, each sample is: labelled protein (swimming lane 1), negative control (transforming swimming lane 2 with the plasmid pNU211R2L5 that does not contain the foreign protein encoding gene), transformant MWPsp-MWPmp6-(His)
6-joint-Met-proinsulin (swimming lane 3), transformant MWPsp-MWPmp8-(His)
6-joint-Met-proinsulin (swimming lane 4), transformant MWPsp-MWPmp9-(His)
6-joint-Met-proinsulin (swimming lane 5), transformant MWPsp-MWPmp10-(His)
6-joint-Met-proinsulin (swimming lane 6), transformant MWPsp-MWPmp11-(His)
6-joint-Met-proinsulin (swimming lane 7), transformant MWPsp-MWPmp12-(His)
6-joint-Met-proinsulin (swimming lane 8), transformant MWPsp-MWPmp15-(His)
6-joint-Met-proinsulin (swimming lane 9), transformant MWPsp-MWPmp40-(His)
6-joint-Met-proinsulin (swimming lane 10), transformant MWPsp-MWPmp50-(His)
6-joint-Met-proinsulin (swimming lane 11), transformant MWPsp-MWPmp100-(His)
6-joint-Met-proinsulin (swimming lane 12).
Fig. 7 is the photo that shows the western blotting result who contains the insulinogenic substratum that does not contain the His-mark that produces by the cultivation transformant, wherein, each sample is: mark peptide (swimming lane 1), negative control (only using plasmid pNU211R2L5 to transform swimming lane 2), transformant MWPsp-proinsulin (swimming lane 3), transformant MWPsp-MWPmp1-Met-proinsulin (swimming lane 4), transformant MWPsp-MWPmp2-Met-proinsulin (swimming lane 5), transformant MWPsp-MWPmp3-Met-proinsulin (swimming lane 6), transformant MWPsp-MWPmp4-Met-proinsulin (swimming lane 7), transformant MWPsp-MWPmp5-Met-proinsulin (swimming lane 8), transformant MWPsp-MWPmp6-Met-proinsulin (swimming lane 9), transformant MWPsp-MWPmp7-Met-proinsulin (swimming lane 10), transformant MWPsp-MWPmp8-Met-proinsulin (swimming lane 11), transformant MWPsp-MWPmp9-Met-proinsulin (swimming lane 12), transformant MWPsp-MWPmp10-Met-proinsulin (swimming lane 13), transformant MWPsp-MWPmp11-Met-proinsulin (swimming lane 14), transformant MWPsp-MWPmp12-Met-proinsulin (swimming lane 15), transformant MWPsp-MWPmp13-Met-proinsulin (swimming lane 16), transformant MWPsp-MWPmp14-Met-proinsulin (swimming lane 17), transformant MWPsp-MWPmp15-Met-proinsulin (swimming lane 18), transformant MWPsp-MWPmp17-Met-proinsulin (swimming lane 19), transformant MWPsp-MWPmp20-Met-proinsulin (swimming lane 20), transformant MWPsp-MWPmp50-Met-proinsulin (swimming lane 21).
Fig. 8 is the photo that shows the electrophoresis result of the substratum that contains the somatostatin that produces by the cultivation transformant, wherein, each sample is: mark peptide (swimming lane 1), transformant MWPsp-somatostatin 28 (swimming lane 2), transformant MWPsp-MWPmp10-(His)
6-EGF-TEV-somatostatin 28 proinsulin (swimming lane 3), transformant MWPsp-MWPmp10-(His)
6-TEV-somatostatin 28 (swimming lane 4) and transformant MWPsp-MWPmp20-(His)
6-EGF-TEV-somatostatin 28 (swimming lane 5).
Fig. 9 is the photo that shows the electrophoresis result of the substratum that contains the hyperglycemic-glycogenolytic factor that produces by the cultivation transformant, and wherein, each sample is: mark peptide (swimming lane 1), transformant MWPsp-hyperglycemic-glycogenolytic factor (swimming lane 2), transformant MWPsp-MWPmp10-(His)
6-joint-V8-hyperglycemic-glycogenolytic factor (swimming lane 3), transformant MWPsp-MWPmp20-(His)
6-joint-V8-hyperglycemic-glycogenolytic factor (swimming lane 4) and transformant MWPsp-MWPmp30-(His)
6-joint-V8-hyperglycemic-glycogenolytic factor (swimming lane 5).
Figure 10 is the photo that shows the electrophoresis/western blotting result who contains the insulinogenic substratum that links to each other with the His-mark that produces by the cultivation transformant, wherein, each sample is: negative control (only using plasmid pNU211R2L5 to transform swimming lane 1), transformant MWPsp-MWPmp6-(His)
6-joint-Met-proinsulin (swimming lane 2), transformant MWPsp-MWPmp8-(His)
6-joint-Met-proinsulin (swimming lane 3), transformant MWPsp-MWPmp9-(His)
6-joint-Met-proinsulin (swimming lane 4), transformant MWPsp-MWPmp10-(His)
6-joint-Met-proinsulin (swimming lane 5), transformant MWPsp-MWPmp11-(His)
6-joint-Met-proinsulin (swimming lane 6), transformant MWPsp-MWPmp12-(His)
6-joint-Met-proinsulin (swimming lane 7), transformant MWPsp-MWPmp15-(His)
6-joint-Met-proinsulin (swimming lane 8), transformant MWPsp-MWPmp40-(His)
6-joint-Met-proinsulin (swimming lane 9), transformant MWPsp-MWPmp50-(His)
6-joint-Met-proinsulin (swimming lane 10), transformant MWPsp-MWPmp100-(His)
6-joint-Met-proinsulin (swimming lane 11).
Figure 11 is the photo that shows the electrophoresis/western blotting result who contains the insulinogenic substratum that does not contain the His-mark that produces by the cultivation transformant, wherein, each sample is: negative control (only using plasmid pNU211R2L5 to transform swimming lane 1), transformant MWPsp-proinsulin (swimming lane 2), transformant MWPsp-MWPmp1-Met-proinsulin (swimming lane 3), transformant MWPsp-MWPmp2-Met-proinsulin (swimming lane 4), transformant MWPsp-MWPmp3-Met-proinsulin (swimming lane 5), transformant MWPsp-MWPmp4-Met-proinsulin (swimming lane 6), transformant MWPsp-MWPmp5-Met-proinsulin (swimming lane 7), transformant MWPsp-MWPmp6-Met-proinsulin (swimming lane 8), transformant MWPsp-MWPmp7-Met-proinsulin (swimming lane 9), transformant MWPsp-MWPmp8-Met-proinsulin (swimming lane 10), transformant MWPsp-MWPmp9-Met-proinsulin (swimming lane 11), transformant MWPsp-MWPmp10-Met-proinsulin (swimming lane 12), transformant MWPsp-MWPmp11-Met-proinsulin (swimming lane 13), transformant MWPsp-MWPmp12-Met-proinsulin (swimming lane 14), transformant MWPsp-MWPmp13-Met-proinsulin (swimming lane 15), transformant MWPsp-MWPmp14-Met-proinsulin (swimming lane 14), transformant MWPsp-MWPmp15-Met-proinsulin (swimming lane 17), transformant MWPsp-MWPmp17-Met-proinsulin (swimming lane 18), transformant MWPsp-MWPmp20-Met-proinsulin (swimming lane 19), transformant MWPsp-MWPmp50-Met-proinsulin (swimming lane 20).
Figure 12 shows through separating the also fusion rotein MWPmp10-(His) of purifying
6-joint-Met-proinsulin and handle and the photo of the cracking insulinogenic electrophoresis result of getting off therefrom by cyanogen bromide, wherein, each sample is: mark peptide (swimming lane 1), separate and the fusion rotein MWPmp10-(His) of purifying
6-joint-Met-proinsulin (30 micrograms, swimming lane 2), cracking is got off from described fusion rotein by the cyanogen bromide processing proinsulin (30 micrograms, swimming lane 3) and proinsulin (Sigma) (2 micrograms, swimming lane 4).
Figure 13 shows through separating the also fusion rotein MWPmp10-(His) of purifying
6-joint-Met-proinsulin and handle and insulinogenic electrophoresis/western blotting result's of getting off of cracking photo therefrom by cyanogen bromide, wherein, each sample is: separate and the fusion rotein MWPmp10-(His) of purifying
6-joint-Met-proinsulin (0.3 microgram, swimming lane 1), cracking is got off from described fusion rotein by the cyanogen bromide processing proinsulin (0.3 microgram, swimming lane 2) and proinsulin (Sigma) (0.3 microgram, swimming lane 3).
Figure 14 shows through separating the also fusion rotein MWPmp20-(His) of purifying
6-EGF-TEV-somatostatin 28 and by the TEV protease treatment electrophoresis/western blotting result's of the somatostatin 28 that gets off of cracking photo therefrom, wherein, each sample is: separate and the fusion rotein MWPmp20-(His) of purifying
6-EGF-TEV-somatostatin 28 (104 micrograms, swimming lane 1; 52 micrograms, swimming lane 3; 26 micrograms, swimming lane 5), cracking is got off from described fusion rotein by the TEV protease treatment somatostatin 28 (104 micrograms, swimming lane 2; 52 micrograms, swimming lane 4; 26 micrograms, swimming lane 6) and somatostatin 28 (BACHEM) (4.5 micrograms, swimming lane 7; 1.5 microgram, swimming lane 8).
Figure 15 shows through separating the also fusion rotein MWPmp20-(His) of purifying
6-joint-V8-hyperglycemic-glycogenolytic factor and by the V8 protease treatment electrophoresis/western blotting result's of the hyperglycemic-glycogenolytic factor that gets off of cracking photo therefrom, wherein, each sample is: separate and the fusion rotein MWPmp20-(His) of purifying
6-joint-V8-hyperglycemic-glycogenolytic factor (90 micrograms, swimming lane 1; 45 micrograms, swimming lane 3; 22.5 microgram, swimming lane 5), cracking is got off from described fusion rotein by the V8 protease treatment hyperglycemic-glycogenolytic factor (90 micrograms, swimming lane 2; 45 micrograms, swimming lane 4; 22.5 microgram, swimming lane 6) and hyperglycemic-glycogenolytic factor (Japanese Shimizu Pharmaceutical Co., Ltd) (1.5 micrograms, swimming lane 7).
Figure 16 shows to be used to estimate the fusion rotein MWPmp10-(His) that is produced
6The electrophoresis of-joint-insulinogenic amount of Met-/western blotting result's photo, wherein, each sample is: by cultivating transformant MWPmp10-(His)
6-joint-Met-proinsulin and the substratum (1 microlitre, the swimming lane 1 that obtain; 1/3 microlitre, swimming lane 2; 1/3
2Microlitre, swimming lane 3; 1/3
3Microlitre, swimming lane 4; 1/3
4Microlitre, swimming lane 5) and proinsulin (Sigma) (1 microlitre, swimming lane 6; 0.3 microlitre, swimming lane 7; 0.1 microlitre, swimming lane 8; 0.03 microlitre, swimming lane 9; 0.01 microlitre, swimming lane 10).
Figure 17 shows the aminoacid sequence of fusion protein product MWPsp-MWPmp20-TEV-G-GH and the nucleotide sequence of this product of coding.
Figure 18 shows the synoptic diagram of fusion product MWPsp-MWPmp20-TEV-G-GH being introduced the mode of bacillus brevis expression vector (pNU211R2L5).
Figure 19 is the photo that shows the electrophoresis result of the substratum that contains the human growth hormone that produces by the cultivation transformant, wherein, each sample is: labelled protein (swimming lane 1), negative control (only using plasmid pNU211R2L5 to transform swimming lane 2), transformant MWPsp-GH (swimming lane 3), transformant MWPsp-TEV-G-GH (swimming lane 4), transformant MWPsp-MWPmp1-TEV-G-GH (swimming lane 5), transformant MWPsp-MWPmp2-TEV-G-GH (swimming lane 6), transformant MWPsp-MWPmp3-TEV-G-GH (swimming lane 7), transformant MWPsp-MWPmp4-TEV-G-GH (swimming lane 8), transformant MWPsp-MWPmp5-TEV-G-GH (swimming lane 9), transformant MWPsp-MWPmp6-TEV-G-GH (swimming lane 10), transformant MWPsp-MWPmp7-TEV-G-GH (swimming lane 11), transformant MWPsp-MWPmp8-TEV-G-GH (swimming lane 12), transformant MWPsp-MWPmp9-TEV-G-GH (swimming lane 13), transformant MWPsp-MWPmp10-TEV-G-GH (swimming lane 14), transformant MWPsp-MWPmp11-TEV-G-GH (swimming lane 15), transformant MWPsp-MWPmp12-TEV-G-GH (swimming lane 16), transformant MWPsp-MWPmp14-TEV-G-GH (swimming lane 17), transformant MWPsp-MWPmp20-TEV-G-GH (swimming lane 18), transformant MWPsp-MWPmp30-TEV-G-GH (swimming lane 19).
Figure 20 is the photo that shows the western blotting result of the substratum that contains the human growth hormone that produces by the cultivation transformant, wherein, each sample is: negative control (only using plasmid pNU211R2L5 to transform swimming lane 1), transformant MWPsp-GH (swimming lane 2), transformant MWPsp-TEV-G-GH (swimming lane 3), transformant MWPsp-MWPmp1-TEV-G-GH (swimming lane 4), transformant MWPsp-MWPmp2-TEV-G-GH (swimming lane 5), transformant MWPsp-MWPmp3-TEV-G-GH (swimming lane 6), transformant MWPsp-MWPmp4-TEV-G-GH (swimming lane 7), transformant MWPsp-MWPmp5-TEV-G-GH (swimming lane 8), transformant MWPsp-MWPmp6-TEV-G-GH (swimming lane 9), transformant MWPsp-MWPmp7-TEV-G-GH (swimming lane 10), transformant MWPsp-MWPmp8-TEV-G-GH (swimming lane 11), transformant MWPsp-MWPmp9-TEV-G-GH (swimming lane 12), transformant MWPsp-MWPmp10-TEV-G-GH (swimming lane 13), transformant MWPsp-MWPmp11-TEV-G-GH (swimming lane 14), transformant MWPsp-MWPmp12-TEV-G-GH (swimming lane 15), transformant MWPsp-MWPmp14-TEV-G-GH (swimming lane 16), transformant MWPsp-MWPmp20-TEV-G-GH (swimming lane 17), transformant MWPsp-MWPmp30-TEV-G-GH (swimming lane 18).
Figure 21 shows through separating and the fusion rotein MWPmp20-TEV-G-GH of purifying and the western blotting result's of the human growth hormone G-GH of the cracking sudden change of getting off the photo therefrom by the TEV protease treatment, wherein, each sample is: fusion rotein MWPmp20-TEV-G-GH (5 micrograms of labelled protein (swimming lane 1), separation and purifying, swimming lane 2), human growth hormone G-GH (5 micrograms of the sudden change that cracking is got off from described fusion rotein by the TEV protease treatment, swimming lane 3) and human growth hormone (Biogenesis) (5 micrograms, swimming lane 4).
Figure 22 shows through separating and the fusion rotein MWPmp20-TEV-G-GH of purifying and the photo of the electrophoresis result of the human growth hormone G-GH of the cracking sudden change of getting off therefrom by the TEV protease treatment, wherein, each sample is: separate also fusion rotein MWPmp20-TEV-G-GH (0.1 microgram of purifying, swimming lane 1), human growth hormone G-GH (0.1 microgram of the sudden change that cracking is got off from described fusion rotein by the TEV protease treatment, swimming lane 2) and human growth hormone (Biogenesis) (0.1 microgram, swimming lane 3).
Detailed description of the present invention
According to the present invention, the polypeptide with required natural type primary structure can be by carrying out chemistry or zymetology and process and obtain expressing fusion that above-mentioned DNA produces in bacillus.
Example from one or more amino acid residues of the CWP protein N terminal of bacillus is that (deposit number is FERM BP-1664 from (but being not limited to) bacillus brevis bacterial strain 47-5Q; JP-A-60-58074; JP-A-62-201589) and bacillus brevis bacterial strain HPD31 (deposit number is FERM BP-1087; JP-A-04-0278091) those residues. For example, can use following sequence:
MWPmp10:Ala Glu Glu Ala Ala Thr Thr Thr Ala Pro (SEQ ID NO:3; Bacteriology magazine 169:1239-1245,1989);
OWPmp10:Ala Pro Lys Asp Gly Ile Tyr Ile Gly Gly (SEQ ID NO:4; Bacteriology magazine 170:176-186,1988);
HWPmp10:Ala Glu Asp Thr Thr Thr Ala Pro Lys Met (SEQ ID NO:5; Bacteriology magazine 172:1312-1320,1990).
Number from the amino acid residue of described N end is generally one or more, is preferably 6 or more, more preferably 6,7,8,10,11,12,13,14,15,17,20 or 50.
Chemistry or enzymatic lysis about amino acid residue, the example of chemical cracking comprises the selective splitting (journal of biological chemistry that is positioned at methionine C-end side, 237:1856-1860,1962) and be positioned at the selective splitting (Enzymology method of tryptophan C-end side, 91:318-324,1983); And the example of enzymatic lysis is by the selective splitting to position of fusion such as factor Xa, fibrin ferment, enterokinase, V8 protease, TEV protease or similar enzyme. Because to the N end side that chemistry or the enzymatic lysis of amino acid residue is positioned at desired polypeptides, chemistry subsequently or enzymatic lysis can cause having the generation of the polypeptide of required primary structure.
In the present invention, allogenic polypeptide derives from the polypeptide of any organism, as long as it does not affect above-mentioned chemistry or enzymatic lysis program. Specifically, when the chemical cracking that adopts chemical cracking particularly to be undertaken by cyanogen bromide, in the primary structure (or amino acid sequence) of purpose allogenic polypeptide, should not contain methionine residues. The example of described polypeptide is growth factor A chain (PDGF-A), people's secretin and the analog etc. that (being not limited to) proinsulin human, human blood platelets are derived. When special employing TEV protease carried out enzymatic lysis, in order to obtain the polypeptide identical with Natural Types, described allogenic polypeptide must have glycine or serine residue at the N end side. The example of these allogenic polypeptides is human somatotropin's inhibin 28, the human blood platelets growth factor A chain (PDGF-A) of deriving, growth factor of human nerve (NGF) etc., yet, the object lesson that described polypeptide is not limited to enumerate above is as long as add the function that glycine or serine do not affect allogenic polypeptide in that N is terminal. When using V8 protease to carry out enzymatic lysis, allogenic polypeptide should never contain glutaminic acid residue. The example of this peptide species is human glucagon, atrial, natriuretic peptides of humans, HCT etc.
According to the present invention, DNA can preferably contain the nucleotide sequence, the particularly nucleotide sequence of MWP signal peptide of the bacillus CWP signal peptide that is coded in fusion N end.
DNA of the present invention also can contain coding as the amino acid whose nucleotide sequence of the mark of separation and purifying and/or the amino acid whose nucleotide sequence that coding is known as joint.
The term used herein mark of purifying " separate and " refers to help peptide that the fusion for preparing by gene recombination technology expression is separated.
Combination between the material of preferred flag sequence and with it combination is reversible. Described flag sequence comprise (for example) and glutathione by the glutathione S-transferase of compatibility, with amylose by the maltose-binding protein of compatibility, wherein histidine and the metal peptide sequence, antigen and the antibody thereof that are formed by the histidine residues of compatibility, etc. In preferred embodiment of the present invention, described flag sequence be His His His His His His (SEQ ID NO:61) (that is, (His)6)。
Joint is present between the interior functional structure territory of protein usually, and has the effect that connects described domain but do not affect the function of described domain. In the present invention, joint for separating of and the flag sequence and allogenic polypeptide of purifying between, and work to make the expressing fusion protein of the flag sequence that band inserts to some extent/secretion. The example of used joint is the combination that is selected from the different numbers of amino acid residue of Ala, Gly, Pro, Ser and Val. In the preferred embodiment of the invention, described joint is Gly Ser Pro Val Pro Ser Gly. Yet, if do not have insertion for separating of the flag sequence that reaches purifying, joint can be mixed in the fusion, can it not mixed yet. Comprise in the situation of somatostatin 28 as allogenic polypeptide at fusion, special joint such as EGF be guarantee allogenic polypeptide expression/secretion is necessary.
In one embodiment of the invention, the invention provides a kind of DNA that comprises the nucleotide sequence of encoding fusion protein, described fusion comprises: by be derived from the MWP protein N terminal one or more, preferred 6-50 (not comprising 9) is individual, particularly the sequence that forms of 6,7,8,9,10,11,12,13,14,15,17,20 or 50 amino acid residues (hereinafter referred is MWPmp6, MWPmp7, MWPmp8, MWPmp10, etc.), by 6 histidine residues form as separate and the sequence of the mark of purifying (in this article with (His)6Expression), amino acid sequence Gly Ser Pro Val Pro Ser Gly as joint, be used for desired polypeptides cracking required methionine residues out, and do not contain the peptide sequence of methionine in its amino acid sequence, wherein said MWP albumen is a kind of of CWP; Described these sequences are according to the order of sequence linear connection each other, and wherein said nucleotide sequence links to each other with 3 ' of the nucleotide sequence that comprises the bacillus promoter district-end. The example of this peptide species is the proinsulin human. Described flag sequence or joint are the elements of choosing wantonly. Described fusion also can further comprise the MWP signal peptide sequence that is positioned at the N end.
In another embodiment, the invention provides a kind of DNA that comprises the nucleotide sequence of encoding fusion protein, described fusion comprises: by be derived from the MWP protein N terminal one or more, preferred 6-50 (not comprising 9) is individual, 10 or 20 sequences that amino acid residue forms particularly, by 6 histidine residues form as separating and the sequence of the mark of purifying, as hEGF's sequence of joint, with TEV protease with desired polypeptides cracking required amino acid sequence Asp Tyr Asp Ile Pro Thr Thr Glu Asn Leu Tyr Phe Gln out, with in its amino acid sequence, do not contain the TEV protease recognition sequence but its N end have glycine or serine peptide sequence, wherein said MWP albumen is a kind of of cell wall protein; Described these sequences are according to the order of sequence linear connection each other, and wherein said nucleotide sequence links to each other with 3 ' of the nucleotide sequence that comprises the bacillus promoter district-end. The example of described polypeptide is somatostatin 28. Described fusion also can further comprise the MWP signal peptide sequence that is positioned at the N end.
In another embodiment, the invention provides a kind of DNA that comprises the nucleotide sequence of encoding fusion protein, described fusion comprises: by be derived from the MWP protein N terminal one or more, preferred 6-50 (not comprising 9) is individual, 20 sequences that amino acid residue forms particularly, by 6 histidine residues form as separating and the sequence of the mark of purifying, amino acid sequence Gly Ser Pro Val Pro Ser Gly as joint, with V8 protease with desired polypeptides cracking required amino acid sequence Phe Leu Glu out, with the peptide sequence that does not contain glutamic acid in its amino acid sequence, wherein said MWP albumen is a kind of of cell wall protein; Described these sequences are according to the order of sequence linear connection each other, and wherein said nucleotide sequence links to each other with 3 ' of the nucleotide sequence that comprises the bacillus promoter district-end. The example of described polypeptide is human glucagon. Described fusion also can further comprise the MWP signal peptide sequence that is positioned at the N end.
According to a further aspect in the invention, the invention still further relates to a kind of DNA that comprises the nucleotide sequence of encoding fusion protein, described fusion comprises: the CWP signal peptide sequence of bacillus, the sequence and the allogenic polypeptide sequence that are comprised of the amino acid residue that is used for enzymatic lysis; Described these sequences are according to the order of sequence linear connection each other, and wherein said nucleotide sequence links to each other with 3 ' of the nucleotide sequence that comprises the bacillus promoter district-end.
Can directly follow described signal peptide sequence by the sequence that the one or more amino acid residues from the CWP protein N terminal form. The sequence that is used for enzymatic lysis can be easily by the sequence of the cracking such as protease such as factor Xa, fibrin ferment, enterokinase, V8 protease or TEV protease.
In another embodiment of the present invention, fusion comprises: the signal peptide sequence of MWP, with TEV protease with desired polypeptides cracking required amino acid sequence Asp Tyr Asp Ile Pro Thr Thr Glu Asn Leu Tyr Phe Gln and in its amino acid sequence, do not contain the peptide sequence of TEV protease recognition sequence out, wherein said MWP albumen is a kind of of cell wall protein; Described these sequences are according to the order of sequence linear connection each other.
In this case, the sequence that is comprised of the one or more amino acid residues from the MWP protein N terminal can directly be followed described signal peptide sequence. If comprise the sequence from the MWP protein N terminal in the fusion, preferably this sequence comprises 1,2,3,4,5,6,7,8,9,10,11,12,14,20 or 30 amino acid. The example of described polypeptide is at the human growth hormone (HGH) of N end for the sudden change of glycine.
In the present invention, the encode nucleotide sequence of above-mentioned fusion links to each other with 3 ' of the nucleotide sequence that comprises the bacillus promoter district-end. Suitable promoter is that (but being not limited to) derives from bacillus brevis bacterial strain 47-5Q (JP-B-01-58950; JP-B-07-108224) MWP promoter and derive from bacillus brevis bacterial strain HPD31 (JP-A-06-278091; JP-A-06-133782) the HWP promoter, etc.
DNA of the present invention can prepare by the combination of known technology of the prior art. For example, the dna sequence dna of each element can prepare respectively by chemical synthesis or clone; In conjunction with pcr amplification (that is, the PCR), the NDA sequence that obtains is coupled together according to the order of sequence with ligase, to obtain target DNA. Reference is described embodiment hereinafter, and its details can be distincter. About can be used for various common technology of the present invention, see also: Maniatis, the people such as T. edit, molecular cloning second edition, laboratory manual, cold spring harbor laboratory (1989); Innis, the people such as N.A., PCR Protocols, methods and applications guide, Academic publishing house (1990).
The DNA of encoding exogenous polypeptide can utilize conventional clone technology and obtain. For example, with the allogenic polypeptide purifying and measure its partial amino-acid series; On the basis of measured sequence, synthesising probing needle or preparation antibody; And screen the cDNA library of containing purpose cDNA with probe or antibody, the DNA of the desired polypeptides that obtains thus encoding. For short DNA, can utilize the imino phosphonate chemistry synthetic at commercially available dna synthesizer. If necessary, can carry out pcr amplification to DNA, wherein with the DNA sex change, with the annealing of primer and extension is cycled to repeat 20 times or more than.
The present invention also provides the carrier that comprises DNA defined above.Can be used for carrier of the present invention and must have the restriction site that DNA maybe can be introduced in suitable insertion site, allow DNA in the genus bacillus host cell, to express, and can be in this host cell self-replicating.The complementary gene that can comprise replication orgin, terminator sequence, ribosome bind site or selectable mark such as drug resistant gene and auxotroph feature at carrier.Carrier of the present invention is preferably plasmid.The example of carrier comprises pUN200, pHY500 (Proc.Natl.Acad.Sci.USA86:3589-3593), pHY4831 (bacteriology magazine 169:1239-1245,1987), pUN100 (applied microbiology biotechnology 30:75-80,1989), pNU211 (biochemical magazine 112:488-491,1992), pNU211R2L5 (JP-A-07-170984), pHY700 (JP-A-04-278091), pHT210 (JP-A-06-133782) and pHT110R2L5 (applied microbiology biotechnology 42:358-363,1994).In the described below example, can prepare expression vector by the construction process shown in Fig. 5 and 18, that is, and pNU-PINS-1, pNU-PINS-2, pNU-STN, pNU-GCN and pNU-G-GH.
The present invention also provides the bacterium that belongs to bacillus that transforms with carrier defined above.Can be used for genus bacillus of the present invention is that (deposit number is FERM BP-1664 to (but being not limited to) bacillus brevis bacterial strain 47-5Q; JP-A-60-58074; JP-A-62-201589), (deposit number is FERM BP-1087 for bacillus brevis bacterial strain 47K (JP-A-02-257876), bacillus brevis bacterial strain 31OK (JP-A-06-296485), bacillus brevis bacterial strain HPD31; JP-A-04-0278091), or the like.Expression vector pNU-PINS-1, the pNU-PINS-2, pNU-STN and the pNU-GCN that are changed over to bacillus brevis bacterial strain 47-5Q are preserved in expert on Japan's business men according to budapest treaty respectively and already economize the Industrial Technology Institute life engineering (1-3 of Industrial Technology Research Institute, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan); Its deposit number is FERM BP-6311, FERM BP-6312, FERM BP-6313 and FERM BP-6314.
The carrier that as above obtains is introduced in the competent bacillus cell, in suitable substratum, bacillus cell is cultivated under the condition that can make vector expression then, thus in cell or the extracellular, preferably produce recombinant fusion polypeptide in the extracellular; And by routine techniques is collected and purifying is produced polypeptide.The example of described introducing carrier is electroporation (Enzymology method 217:23-33,1993).Can adopt suitable mixed gel filtration, ion-exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, electrophoresis or the like that the fusion rotein that is obtained is carried out purifying.
Can carry out chemistry or enzymatic lysis to fusion rotein subsequently, to obtain having the desired polypeptides of natural primary structure.Handle about cracking, can adopt the chemical cracking of methionine(Met) or tryptophane C-end side and the enzymatic lysis that is undertaken by factor Xa, zymoplasm, enteropeptidase, V8 proteolytic enzyme or TEV proteolytic enzyme.
The present invention also provides a kind of method for preparing recombinant polypeptide, and this method comprises: cultivate the bacterium that belongs to bacillus that is as above transformed in substratum, with the extracellular on this bacterium the fusion rotein that comprises allogenic polypeptide is gathered; From substratum, isolate fusion rotein; From the isolated fusion rotein of institute, described allogenic polypeptide cracking is come out; With the described polypeptide of collection.
The recombinant polypeptide of preparing by this method of the present invention can be used for pharmacy, diagnosis, scientific research or the like.
Embodiment
Hereinafter will be described in more detail the present invention by embodiment in conjunction with the accompanying drawings.
In an embodiment, fusion rotein prepares as follows: with the normal chain and the annealing of minus strand oligonucleotide of chemosynthesis; Adopt oligonucleotide by PCR reaction (polymerase chain reaction) amplification of DNA fragments; Adopt dna ligase the dna fragmentation that amplifies to be coupled together by ligation.In this article, " MWPsp " is meant the proteic signal peptide of MWP, follows numeral in the MWPmp back (as, MWPmp1,2,3 ...) be meant amino acid whose number from MWP maturation protein N-end (as, 1,2,3 ... amino acid).
Structure mixes fusion dna MWPsp-MWPmp10-(His)
6-
The insulinogenic carrier pPINS-1 of joint-Met-
(1) preparation dna fragmentation MWPsp-MWPmp10
Following (i) to (iv) adding in the 0.5ml test tube, obtained 100 μ l reaction solns, according to currently known methods (Innis, M.A. etc., PCR introduction, methods and applications guide, Academic Press, 1990) carry out the PCR reaction, repeat 30 circulations: 94 ℃ of sex change 1 minute; Annealed 1 minute for 55 ℃; 72 ℃ prolong DNA chain 1 minute.
(i) template DNA
The 840ng genomic dna is to extract from bacillus brevis (bacterial strain 47-5Q) according to currently known methods (molecular cloning second edition, laboratory manual, cold spring harbor laboratory (1989)).
(ii) primer
Forward primer 5 '-GTCGTTAACAGTGTATTGCT-3 ' (SEQ ID NO:6) and reverse primer 5 '-TGGAGCTGTAGTAGTTGCTGCTTCTTCTGC-3 ' (SEQ IDNO:7), be with Yamagata, (bacteriology magazine 169 such as H., 1239-1245,1987) and proteinic nucleotides sequence of MWP of determining of Tsuboi.A. etc. (bacteriology magazine 170,935-945,1988) classify the basis as, with organic synthesis method preparation, these primers are added to the final concentration of 0.1 μ M.
(iii) Taq DNA synthetic enzyme
The commercially available Taq DNA of 5U synthetic enzyme (GIBCO BRL).
(iv) other
Tris-HCl (final concentration is 20mM, pH8), and MgCl
2(final concentration is 2.5mM) and dNTPs (dATP, dGTP, dCTP and dTTP, each 50 μ M of final concentration).
When the PCR reaction finishes, use the phenol concentrated reaction mixture, add to then in 0.8% sepharose, under normal operation, carry out electrophoresis.From sepharose, reclaim PCR product, i.e. dna fragmentation MWPsp-MWPmp10 with Ultrafree C3H (MolliporeCorp.).The PCR product that reclaims is handled with phenol, and through ethanol sedimentation, vacuum-drying is dissolved in an amount of distilled water then.After this, by the explanation of manufacturers, (Takara Shuzo, Co. Ltd) make blunt end with DNA Blunting test kit with gained PCR product.
(2) preparation dna fragmentation (His)
6
According to genetic code table (molecular cloning second edition, laboratory manual, cold spring harbor laboratory (1989)), chemical synthesis coding (His)
6Forward oligonucleotide 5 '-CATCATCATCATCATCAC-3 ' (SEQ ID NO:8) and reverse oligonucleotide 5 '-GTGATGATGATGATGATG-3 ' (SEQ ID NO:9).By the explanation of manufacturers, use T4 polynucleotide kinase (Nippon Gene) with described oligonucleotide phosphorylation, in 95 ℃, at 10mM Tris-HCl (pH8) and 5mM MgCl
2Handled 5 minutes in the solution, and 37 ℃ of annealing 15 minutes.With annealed double chain DNA fragment (His)
6Handle with phenol, through ethanol sedimentation, vacuum-drying is dissolved in an amount of distilled water then.
(3) preparation dna fragmentation joint
According to genetic code table (with above), the forward oligonucleotide 5 of chemical synthesis coding joint Gly Ser Pro Val Pro SerGly (SEQ ID NO:1) '-GGTTCTCCAGTACCTTCTGGA-3 ' (SEQ ID NO:53) and reverse oligonucleotide 5 '-TCCAGAAGGTACTGGAGAACC-3 ' (SEQ ID NO:10), anneal to obtain the double chain DNA fragment joint according to described in the present embodiment (2) then.
(4) preparation dna fragmentation proinsulin
According to identical method described in the present embodiment (1), the fragment proinsulin of preparation blunt end DNA, different is:
(a) make template DNA with the plasmid vector 10ng that mixes proinsulin human DNA, this carrier prepares by following method: according to the explanation of manufacturers, with the first chain cDNA synthetic agent box (Pharmacia), from the synthetic human pancreas cDNA of commercially available human pancreas mRNA (Clontech); With Bell, the nucleotides sequence of the human proinsulin gene that G.I. etc. (Nature, 282,525-527,1979) determine is classified the basis as, synthetic forward primer 5 '-ATGGCCCTGTGGATGCGCC-3 ' (SEQID NO; 11) and reverse primer 5 '-CTAGTTGCAGTAGTTCTCC-3 ' (SEQ IDNO:12); Carry out the PCR reaction with above-mentioned cDNA and the synthetic oligonucleotide that obtains, repeat 35 circulations: handled 1 minute at 94 ℃, handled 1 minute for 60 ℃, handled 1 minute for 72 ℃ as template; With the PCR product that obtains thus, promptly proinsulin human's dna clone is in pGEM-T carrier (Promega);
(b) use forward primer 5 '-TTTGTGAACCAACACCTG-3 ' (SEQ IDNO:13) and reverse primer 5 '-CTAGTTGCAGTAGTTCTCC-3 ' (SEQ IDNO:12); With
(c) by repeating 25 circulations: 94 ℃ of sex change 1 minute, 53 ℃ of annealing 1 minute, 72 ℃ prolong 30 seconds of DNA chain; Finish the PCR reaction.
(5) preparation dna fragmentation Met-proinsulin
According to identical method described in the present embodiment (4), preparation blunt end dna fragmentation Met-proinsulin, difference is: (a) with the 10ng PCR product Regular Insulin original work template DNA that obtains in the present embodiment (4); (b) use forward primer 5 '-ATGTTTGTGAACCAACACCTG-3 ' (SEQ ID NO:54).
According to the explanation of manufacturers, with T4 polynucleotide kinase (Nippon Gene), make blunt end dna fragmentation Met-proinsulin again through phosphorylation reaction, obtain phosphorylated cdna fragment Met-proinsulin thus.
(6) preparation fusion dna MWPsp-MWPmp10-(His)
6
According to identical method described in the present embodiment (1), preparation blunt end fusion dna MWPsp-MWPmp10-(His)
6, different is: (a) with dna ligation kit (TakaraShuzo, Co., Ltd.), by an amount of dna fragmentation MWPsp-MWPmp10 that will obtain in the present embodiment (1) an amount of dna fragmentation (His) with the middle gained of present embodiment (2)
6Prepared template DNA in 30 minutes 16 ℃ of reactions; (b) use reverse primer 5 '-GTGATGATGATGATGATG-3 ' (SEQ ID NO:9); (c) repeat 25 circulations: 94 ℃ of sex change 1 minute, 45 ℃ of annealing 1 minute, 72 ℃ with DNA chain extension 30 seconds; Finish the PCR reaction.
After this, according to the explanation of manufacturers, with T4 polynucleotide kinase (Nippon Gene), with the PCR product phosphorylation of gained.With dna ligation kit (Takara Shuzo, Co., Ltd.) the PCR product with phosphorylation imports carrier (the BlueScript SK-that HincII cuts, Stratagene) in, so that with currently known methods (molecular cloning second edition, laboratory manual, cold spring harbor laboratory (1989)) transformed into escherichia coli DH5 α.Plasmid purification carrier DNA from transformant.In order to determine whether to have obtained fusion dna MWPsp-MWPmp10-(His)
6,, determine the nucleotide sequence of carrier with the primer forward or backwards (being M13 primer forward or backwards) of sequencing vector.According to method same as described above, finish the 2nd PCR reaction, with mixing MWPsp-MWPmp10-(His)
6Carrier make template DNA, and use forward primer 5 '-GTCGTTAACAGTGTATTGCT-3 ' (SEQ ID NO:6) and reverse primer 5 '-GTGATGATGATGATGATG-3 ' (SEQ ID NO:9), thus, the fusion dna MWPsp-MWPmp10-(His) of preparation blunt end
6
(7) preparation fusion dna MWPsp-MWPmp10-(His)
6-joint
According to preparing blunt end fusion dna MWPsp-MWPmp10-(His) with the described identical method of present embodiment (6)
6-joint, different is: (a) (Takara Shuzo, Co. is Ltd.) by an amount of fusion dna MWPsp-MWPmp10-(His) with above-mentioned (6) middle gained with dna ligation kit
6Prepared the template DNA that is used for PCR reaction with an amount of dna fragmentation joint that obtains in above-mentioned (3) in 30 minutes 16 ℃ of reactions; (b) reverse primer 5-TCCAGAAGGTACTGGAGAACC-3 ' (SEQ ID NO:10) is used for PCR reaction.
(8) preparation is mixed with fusion dna MWPsp-MWPmp10-(His)
6The insulinogenic carrier of-joint-Met-
According to mixing fusion product MWPsp-MWPmp10-(His) with method preparation identical described in the present embodiment (6)
6The insulinogenic carrier pPINS-1 of-joint-Met-, different is (a) with dna ligation kit (Takara Shuzo, Co., Ltd.), by with gained fusion dna MWPsp-MWPmp10-(His) in an amount of present embodiment (7)
6-joint prepared the template DNA that is used for PCR reaction with an amount of present embodiment (5) gained dna fragmentation Met-proinsulin in 30 minutes 16 ℃ of reactions; (b) reverse primer 5 '-CTAGTTGCAGTAGTTCTCC-3 ' (SEQID NO:12) is used for PCR reaction.
Make up respectively and be mixed with fusion dna MWPsp-MWPmp6-, 8-, 9-, 11-, 12-, 15-, 40-, 50-, 100-(His)
6The insulinogenic carrier of-joint-Met-
(1) preparation dna fragmentation MWPsp-MWPmp6,8,9,11,12,15,40,50,100
According to embodiment 1 (1) described identical method, preparation dna fragmentation MWPsp-MWPmp6,8,9,11,12,15,40,50,100, different is:
(a) with following primer as reverse primer:
MWPmp6：5′-AGTTGCTGCTTCTTCTGC-3′(SEQ ID NO：14)
MWPmp8：5′-TGTAGTAGTTGCTGCTTC-3′(SEQ ID NO：15)
MWPmp9：5′-AGCTGTAGTAGTTGCTGC-3′(SEQ ID NO：16)
MWPmp11：5′-TTTTGGAGCTGTAGTAGT-3′(SEQ ID NO：17)
MWPmp12：5′-CATTTTTGGAGCTGTAGT-3′(SEQ ID NO：18)
MWPmp15：5′-ATCAGCGTCCATTTTTGG-3′(SEQ ID NO：19)
MWPmp40：5′-GTCTACACCGTATTCGCCGT-3′(SEQ ID NO：20)
MWPmp50：5′-AGTAGCGAACTCTGCACGAG-3′(SEQ ID NO：21)
MWPmp100：5′-AGATTTGTCCGGGAAACCTT-3′(SEQ ID NO：22)；
(b) by repeating 30 circulations: 94 ℃ of sex change 1 minute, 45 ℃ of annealing 1 minute, 72 ℃ prolong DNA chain 1 minute; To finish the PCR reaction.
(2) preparation dna fragmentation (His)
6-joint-Met-proinsulin
According to identical method described in the embodiment 1 (1), the preparation blunt end dna fragmentation (His)
6-joint-Met-proinsulin, what different was (a) with gained among the 10ng embodiment 1 (8) is mixed with fusion dna MWPsp-MWPmp10-(His)
6The carrier pPINS-1 of-joint is as template DNA; (b) use forward primer 5 '-CATCATCATCATCATCAC-3 ' (SEQ IDNO:8) and reverse primer 5 '-CTAGTTGCAGTAGTTCTC-3 ' (SEQ ID NO:23); (c) repeat 25 circulations: 94 ℃ of sex change 1 minute, 47 ℃ of annealing 1 minute, 72 ℃ prolong DNA chain 30 seconds; To finish the PCR reaction.
According to the explanation of manufacturers, with T4 polynucleotide kinase (Nippon Gene), with blunt end dna fragmentation (His)
6-joint-Met-proinsulin is carried out phosphorylation reaction, obtains the dna fragmentation (His) of phosphorylation thus
6-joint-Met-proinsulin.
(3) preparation is mixed with fusion dna MWPsp-MWPmp6-, 8-, 9-, 11-, 12-, 15-, 40-, 50-, 100-(His) respectively
6The insulinogenic carrier of-joint-Met-
Described in embodiment 1 (8), preparation is mixed with fusion dna MWPsp-MWPmp6-, 8-, 9-, 11-, 12-, 15-, 40-, 50-, 100-(His) respectively
6The insulinogenic carrier of-joint-Met-, different is, with dna ligation kit (Takara Shuzo, Co., Ltd.) by respectively with an amount of dna fragmentation MWPsp-MWPmp6-of preparation in the present embodiment (1), 8-, 9-, 11-, 12-, 15-, 40-, 50-, 100 with an amount of dna fragmentation (His) of the middle gained of present embodiment (2)
6-joint-Met-proinsulin prepares the template DNA that is used for PCR reaction 16 ℃ of reactions 30 minutes.
Structure is mixed with the insulinogenic carrier of fusion dna MWPsp-MWPmp10-Met-
According to being mixed with the insulinogenic carrier pPINS-2 of fusion dna MWPsp-MWPmp10-Met-with embodiment 1 (8) described identical method preparation, different is, with dna ligation kit (Takara Shuzo, Co., Ltd.), the an amount of dna fragmentation Met-proinsulin that obtains by an amount of dna fragmentation MWPsp-MWPmp10 that will obtain among the embodiment 1 (1) and embodiment 1 (5) prepares the template DNA that is used for PCR reaction 16 ℃ of reactions 30 minutes.
Make up and be mixed with fusion dna MWPsp-MWPmp1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 11-, 12-, 13-, 14-, 15-, 17-, 20-, the insulinogenic carrier of 50-Met-respectively
(1) preparation dna fragmentation MWPsp-MWPmp1,2,3,4,5,7,13,14,17,20
According to preparing blunt end dna fragmentation MWPsp-MWPmp1,2,3,4,5,7,13,14,17,20 with embodiment 1 described identical method, different is:
(a) with following primer as reverse primer:
MWPmp1：5′-TGCTGCGAAAGCCATTGG-3′(SEQ ID NO：24)
MWPmp2：5′-TTCTGCTGCGAAAGCCAT-3′(SEQ ID NO：25)
MWPmp3：5′-TTCTTCTGCTGCGAAAGC-3′(SEQ ID NO：26)
MWPmp4：5′-TGCTTCTTCTGCTGCGAA-3′(SEQ ID NO：27)
MWPmp5：5′-TGCTGCTTCTTCTGCTGC-3′(SEQ ID NO：28)
MWPmp7：5′-AGTAGTTGCTGCTTCTTC-3′(SEQ ID NO：29)
MWPmp13：5′-GTCCATTTTTGGAGCTGT-3′(SEQ ID NO：30)
MWPmp14：5′-AGCGTCCATTTTTGGAGC-3′(SEQ ID NO：31)
MWPmp17：5′-TTCCATATCAGCGTCCAT-3′(SEQ ID NO：32)
MWPmp20：5′-TACGGTTTTTTCCATATCAGC-3′(SEQ ID NO：33)；
(b) repeat 30 circulations: 94 ℃ of sex change 1 minute, 45 ℃ of annealing 1 minute, 72 ℃ prolong DNA chain 1 minute; Finish the PCR reaction.
(2) preparation is mixed with fusion dna MWPsp-MWPmp1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 11-, 12-, 13-, 14-, 15-, 17-, 20-, the insulinogenic carrier of 50-Met-respectively
According to embodiment 1 (8) described identical method, preparation is mixed with fusion dna MWPsp-MWPmp1-respectively, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 11-, 12-, 13-, 14-, 15-, 17-, 20-, the insulinogenic carrier of 50-Met-, different is: (a) with dna ligation kit (Takara Shuzo, Co., Ltd.), by respectively with the dna fragmentation MWPsp-MWPmp1 of gained in an amount of embodiment 2 (1) and the present embodiment (1), 2,3,4,5,6,7,8,9,11,12,13,14,15,17,20,50 with embodiment 1 (5) in an amount of dna fragmentation Met-proinsulin of gained 16 ℃ of reactions 30 minutes, prepare the template DNA that is used for PCR reaction.
Structure is mixed with the insulinogenic carrier of fusion dna MWPsp-
(1) preparation dna fragmentation MWPsp
According to embodiment 1 (1) described identical method, preparation blunt end dna fragmentation MWPsp, different is that reverse primer 5 '-TGCGAAAGCCATTGGAGCAAC-3 ' (SEQ ID NO:34) is used for the PCR reaction.
(2) preparation is mixed with the insulinogenic carrier of fusion dna MWPsp-
According to embodiment 1 (8) described identical method, preparation is mixed with the insulinogenic carrier of fusion dna MWPsp-, different is, with dna ligation kit (Takara Shuzo, Co., Ltd.), by an amount of dna fragmentation MWPsp of gained in the present embodiment (1) and the blunt end dna fragmentation proinsulin of embodiment 1 (4) middle gained were reacted 30 minutes at 16 ℃, prepare the template DNA that is used for PCR reaction.
Make up and be mixed with fusion dna MWPsp-somatostatin 28, MWPsp-MWP10-(His) respectively
6-EGF-TEV-somatostatin 28, MWPsp-MWP10-(His)
6-TEV-somatostatin 28 and MWPsp-MWP20-(His)
6The carrier of-EGF-TEV-somatostatin 28
(1) preparation dna fragmentation somatostatin 28
According to embodiment 1 (1) described identical method, the preparation blunt end dna fragmentation somatostatin 28, different is:
(a) with Shen, (Proc.Natl.Acad.Sci.U.S.A such as L.-P, 79,4575-4579,1982) nucleotides sequence of Que Dinging is classified the basis as, is equipped with 10ng human somatotropin statin 28 single stranded DNAs template DNA (5 '-TCTGCTAACTCAAACCCGGCTATGGCACCCCGAGAACGCAAAGCTGGCTGCAAGAA TTTCTTCTGGAAGACTTTCACATCCTGTTAG-3 ' (SEQ ID NO:55)) with the organic synthesis legal system;
(b) use forward primer 5 '-TCTGCTAACTCAAACCCG-3 ' (SEQ IDNO:35) and reverse primer 5 '-CTAACAGGATGTGAAAGTCTT-3 ' (SEQ IDNO:36); With
(c) repeat 25 circulations: 94 ℃ of sex change 1 minute, 50 ℃ of annealing 1 minute, 72 ℃ prolong DNA chain 10 seconds; Finish the PCR reaction.
By the explanation of manufacturers, (Nippon Gene) carries out phosphorylation reaction with blunt end dna fragmentation somatostatin 28 with the T4 polynucleotide kinase, obtains the dna fragmentation somatostatin 28 of phosphorylation thus.
(2) preparation dna fragmentation EGF
According to embodiment 1 (1) described identical method, preparation blunt end dna fragmentation EGF, different is:
(a) with 10ng human epidermal growth factor (EGF) single stranded DNA (5 '-AACTCTGACTCCGAATGCCCGCTGTCTCACGACGGTTATTGCCTGCATGATGGTGT TTGTATGTATATCGAAGCTCTGGACAAATATGCTTGCAACTGTGTTGTTGGTTACA TCGGTGAGCGTTGCCAGTATCGCGACCTGAAATGGTGGGAACTGCGT-3 ') (SEQ ID NO:56)) as template DNA, this single stranded DNA is with Bell, (nucleic acids research such as G.I., 14,8427-8446,1986) human epidermal growth factor's of Que Dinging nucleotides sequence is classified the basis as, prepares by the organic synthesis method;
(b) use forward primer 5 '-AACTCTGACTCCGAATGC-3 ' (SEQ IDNO:37) and reverse primer 5 '-ACGCAGTTCCCACCATTT-3 ' (SEQ ID NO:38); With
(c) repeat 25 circulations: 94 ℃ of sex change 1 minute, 50 ℃ of annealing 1 minute, 72 ℃ prolong DNA chain 15 seconds; Finish the PCR reaction.
By the explanation of manufacturers, (Nippon Gene) carries out phosphorylation reaction with blunt end dna fragmentation EGF with the T4 polynucleotide kinase, obtains the dna fragmentation EGF of phosphorylation thus.
(3) preparation dna fragmentation TEV
According to genetic code table (with above), chemical synthesis coding by the forward oligonucleotide 5 of the aminoacid sequence of TEV proteolytic enzyme identification '-GACTATGATATCCCGACCACTGAAAACCTGTACTTCCAA-3 ' (SEQ ID NO:57) and reverse oligonucleotide 5 '-TTGGAAGTACAGGTTTTCAGTGGTCGGGATATCATAGTC-3 ' (SEQ IDNO:58), and, obtain double chain DNA fragment TEV thus according to embodiment 1 (2) described annealing.
(4) preparation fusion dna MWPsp-MWPmp10-(His)
6-EGF
Described according to embodiment 1 (6), the fusion dna MWPsp-MWPmp10-(His) of preparation blunt end
6-EGF, different is: (a) with dna ligation kit (Takara Shuzo, Co., Ltd.), with an amount of fusion dna MWPsp-MWPmp10-(His) of gained among the embodiment 1 (6)
6Reacted 30 minutes at 16 ℃ with an amount of dna fragmentation EGF of gained in the present embodiment (2), prepare the template DNA that is used for PCR reaction; (b) reverse primer 5 '-ACGCAGTTCCCACCATTT-3 ' (SEQ ID NO:38) is used for PCR reaction.
(5) preparation fusion dna MWPsp-MWPmp10-(His)
6-TEV
Described according to embodiment 1 (6), the fusion dna MWPsp-MWPmp10-(His) of preparation blunt end
6-TEV, different is: (a) with dna ligation kit (TakaraShuzo, Co., Ltd.), at 16 ℃, by an amount of fusion dna MWPsp-MWPmp10-(His) with gained among the embodiment 1 (6)
6Reacted 30 minutes with an amount of dna fragmentation TEV of gained in the present embodiment (3), preparation is used for the template DNA of PCR reaction; (b) reverse primer 5 ' TTGGAAGTACAGGTTTTC-3 ' (SEQ ID NO:39) is used for PCR reaction.
(6) preparation fusion dna MWPsp-MWPmp 10-(His)
6-EGF-TEV
According to embodiment 1 (6) described identical method, the preparation blunt end fusion dna MWPsp-MWPmp10-(His)
6-EGF-TEV, different is: (a) with dna ligation kit (Takara Shuzo, Co., Ltd.), at 16 ℃, by an amount of fusion dna MWPsp-MWPmp10-(His) with gained in the present embodiment (4)
6An amount of dna fragmentation TEV of gained reacted 30 minutes in-EGF and the present embodiment (3), prepared the template DNA of PCR reaction; (b) reverse primer 5 ' TTGGAAGTACAGGTTTTC-3 ' (SEQ ID NO:39) is used for PCR reaction.
(7) preparation fusion dna MWPsp-MWPmp20-(His)
6
According to identical method described in the embodiment 1 (6), the preparation blunt end fusion dna MWPsp-MWPmp20-(His)
6, different is: (a) with dna ligation kit (TakaraShuzo, Co., Ltd.), at 16 ℃, by with an amount of fusion dna MWPsp-MWPmp20 of gained among the embodiment 4 (1) and an amount of dna fragmentation (His) of embodiment 1 (2) middle gained
6Reacted 30 minutes, and prepared the template DNA that is used for PCR reaction.
(8) preparation fusion dna MWPsp-MWPmp20-(His)
6-EGF
According to identical method described in the embodiment 1 (6), the preparation blunt end fusion dna MWPsp-MWPmp20-(His)
6-EGF, different is: (a) with dna ligation kit (Takara Shuzo, Co., Ltd.), at 16 ℃, by with an amount of fusion dna MWPsp-MWPmp20-of gained (His) in the present embodiment (7)
6Reacted 30 minutes with an amount of dna fragmentation EGF of (2) gained, prepare the template DNA that is used for PCR reaction; (b) reverse primer 5 '-ACGCAGTTCCCACCATTT-3 ' (SEQ ID NO:38) is used for PCR reaction.
(9) preparation fusion dna MWPsp-MWPmp20-(His)
6-EGF-TEV
According to identical method described in the embodiment 1 (6), the preparation blunt end fusion dna MWPsp-MWPmp20-(His)
6-EGF-TEV, different is: (a) with dna ligation kit (Takara Shuzo, Co., Ltd.), at 16 ℃, by with an amount of fusion dna MWPsp-MWPmp20-of gained (His) in the present embodiment (8)
6An amount of dna fragmentation TEV reaction of-EGF and present embodiment (3) gained 30 minutes prepares the template DNA that is used for PCR reaction; (b) reverse primer 5 '-TTGGAAGTACAGGTTTTC-3 ' (SEQ ID NO:39) is used for PCR reaction.
(10) preparation is mixed with fusion dna MWPsp-somatostatin 28, MWPsp-MWPmp10-(His) respectively
6-EGF-TEV-somatostatin 28, MWPsp-MWPmp10-(His)
6-TEV-somatostatin 28 and MWPsp-MWPmp20-(His)
6The carrier of-EGF-TEV-somatostatin 28
According to embodiment 1 (8) described identical method, preparation is mixed with fusion dna MWPsp-somatostatin 28, MWPsp-MWPmp10-(His) respectively
6-EGF-TEV-somatostatin 28, MWPsp-MWPmp10-(His)
6-TEV-somatostatin 28 and MWPsp-MWPmp20-(His)
6The carrier of-EGF-TEV-somatostatin 28, different is: (a) with dna ligation kit (Takara Shuzo, Co., Ltd.), at 16 ℃, by the dna fragmentation MWPsp of gained among the embodiment 5 (1) and the dna fragmentation somatostatin 28 of the middle gained of present embodiment (1) were reacted 30 minutes, prepare the template DNA of the PCR reaction that is used for MWPsp-somatostatin 28; And with dna ligation kit (Takara Shuzo, Co., Ltd.), at 16 ℃, by with an amount of dna fragmentation somatostatin 28 respectively with present embodiment (5), (6) and (9) in an amount of fusion dna MWPsp-MWPmp10-(His) of gained
6-TEV, MWPsp-MWPmp10-(His)
6-EGF-TEV and MWPsp-MWPmp20-(His)
6-EGF-TEV reaction 30 minutes, preparation is used for MWPsp-MWPmp10-(His)
6-EGF-TEV-somatostatin 28, MWPsp-MWPmp10-(His)
6-TEV-somatostatin 28 and MWPsp-MWPmp20-(His)
6The template DNA of the one PCR reaction of-EGF-TEV-somatostatin 28; Reverse primer 5 '-CTAACAGGATGTGAAAGTCTT-3 ' (SEQ ID NO:36) is used for PCR reaction.
Make up and be mixed with fusion dna MWPsp-hyperglycemic-glycogenolytic factor, MWPsp-MWPmp10-(His) respectively
6-joint-V8-hyperglycemic-glycogenolytic factor, MWPsp-MWPmp20-(His)
6-joint-V8-hyperglycemic-glycogenolytic factor (pGCN) and MWPsp-MWPmp30-(His)
6The carrier of-joint-V8-hyperglycemic-glycogenolytic factor
(1) preparation dna fragmentation hyperglycemic-glycogenolytic factor
According to embodiment 1 (1) described identical method, the preparation blunt end the dna fragmentation hyperglycemic-glycogenolytic factor, different is:
(a) with 10ng Porcine glucagon single stranded DNA (5 '-CACAGCCAAGGTACTTTCACATCCGACTACTCTAAATATCTGGATTCCCGTCGCGC TCAAGATTTCGTTCAATGGCTGATGAACACT-3 ' (SEQ ID NO:59)) as template DNA, this single stranded DNA is with Drucher, (journal of biological chemistry such as D.J, 263,13475-13478,1988) the Porcine glucagon nucleotides sequence of Que Dinging is classified the basis as, prepares by the organic synthesis method;
(b) use forward primer 5 '-CACAGCCAAGGTACTTTC-3 ' (SEQ IDNO:40) and reverse primer 5 '-TTAAGTGTTCATCAGCCATTG-3 ' (SEQ IDNO:41); With
(c) repeat 25 circulations: 94 ℃ of sex change 1 minute, 50 ℃ of annealing 1 minute, 72 ℃ prolong DNA chain 10 seconds; Finish the PCR reaction.
By the explanation of manufacturers, (Nippon Gene) carries out phosphorylation reaction with blunt end dna fragmentation hyperglycemic-glycogenolytic factor with the T4 polynucleotide kinase, obtains the dna fragmentation hyperglycemic-glycogenolytic factor of phosphorylation thus.
(2) preparation dna fragmentation V8-hyperglycemic-glycogenolytic factor
According to embodiment 1 (1) described identical method, preparation blunt end dna fragmentation V8-hyperglycemic-glycogenolytic factor, different is:
(a) be template DNA with the Porcine glucagon DNA that obtains in the 10ng present embodiment (1);
(b) use forward primer 5 '-TTCCTGGAACACAGCCAA-3 ' (SEQ IDNO:42) and reverse primer 5 '-TTAAGTGTTCATCAGCCATTG-3 ' (SEQ IDNO:41); With
(c) repeat 25 circulations: 94 ℃ of sex change 1 minute, 50 ℃ of annealing 1 minute, 72 ℃ prolong DNA chain 10 seconds; Finish the PCR reaction.
By the explanation of manufacturers, with T4 polynucleotide kinase (Nippon Gene) blunt end dna fragmentation V8-hyperglycemic-glycogenolytic factor is carried out phosphorylation reaction, obtain the dna fragmentation V8-hyperglycemic-glycogenolytic factor of phosphorylation thus.
(3) preparation dna fragmentation MWPsp-MWPmp30
According to embodiment 1 (6) described identical method, the fusion dna fragment MWPsp-MWPmp30 of preparation blunt end, different is, uses reverse primer 5 '-TGCTACCAGGCCAAGAGCTT-3 ' (SEQ ID NO:43).
(4) preparation fusion dna MWPsp-MWPmp30-(His)
6
According to embodiment 1 (6) described identical method, the preparation blunt end fusion dna fragment MWPsp-MWPmp30-(His)
6, different is, with dna ligation kit (TakaraShuzo, Co., Ltd.), at 16 ℃, by with an amount of dna fragmentation MWPsp-MWPmp30 of gained in the present embodiment (3) and an amount of dna fragmentation (His) of embodiment 1 (2) middle gained
6Reacted 30 minutes, preparation is used for the template DNA of PCR reaction.
(5) preparation fusion dna MWPsp-MWPmp20-, 30-(His)
6-joint
According to embodiment 1 (6) described identical method, the preparation blunt end fusion dna MWPsp-MWPmp20-, 30-(His)
6-joint, different is: (a) with dna ligation kit (Takara Shuzo, Co., Ltd.), at 16 ℃, by with an amount of dna fragmentation joint of gained among the embodiment 1 (3) respectively with an amount of fusion dna MWPsp-MWPmp20-(His) of embodiment 6 (7) and the middle gained of present embodiment (4)
6And MWPsp-MWPmp30-(His)
6Reacted 30 minutes, preparation is used for the template DNA of PCR reaction; (b) in PCR reaction, use reverse primer 5 '-TCCAGAAGGTACTGGAGAACC-3 ' (SEQ IDNO:10).
(6) preparation is mixed with fusion dna MWPsp-hyperglycemic-glycogenolytic factor and MWPsp-MWPmp10-, 20-, 30-(His) respectively
6The carrier of-joint-V8-hyperglycemic-glycogenolytic factor
According to embodiment 1 (8) described identical method, preparation is mixed with fusion dna MWPsp-hyperglycemic-glycogenolytic factor and MWPsp-MWPmp10-, 20-, 30-(His) respectively
6The carrier of-joint-V8-hyperglycemic-glycogenolytic factor, different is: (a) with dna ligation kit (Takara Shuzo, Co., Ltd.), at 16 ℃, by an amount of dna fragmentation MWPsp of gained among the embodiment 5 (1) and an amount of dna fragmentation hyperglycemic-glycogenolytic factor of the middle gained of present embodiment (1) were reacted 30 minutes, preparation is used for the template DNA of the PCR reaction of MWPsp-hyperglycemic-glycogenolytic factor; And with dna ligation kit (Takara Shuzo, Co., Ltd.), at 16 ℃, by with an amount of dna fragmentation V8-hyperglycemic-glycogenolytic factor of gained in the present embodiment (2) respectively with an amount of fusion dna MWPsp-MWPmp10-(His) of embodiment 1 (7) and the middle gained of present embodiment (5)
6-joint and MWPsp-MWPmp20-, 30-(His)
6-joint reaction 30 minutes, preparation is used for MWPsp-MWPmp10-, 20-, 30-(His)
6The template DNA of the one PCR reaction of-joint-V8-hyperglycemic-glycogenolytic factor; (b) reverse primer 5 '-TTAAGTGTTCATCAGCCATTG-3 ' (SEQID NO:41) is used for PCR reaction.
Expression/the secretion of fusion dna and product selectivity cracking
(1) nucleotide sequence of the aminoacid sequence of fusion product and encode such amino acid sequences
In embodiment 1-7, in the fusion product of gained, in SEQ ID NOS:48-51,62-65 and Fig. 1-4, listed the nucleotide sequence and the aminoacid sequence of following product respectively.
MWPsp-MWPmp10-(His)
6-joint-Met-proinsulin
(SEQ ID NO：48，62)
The MWPsp-MWPmp10-Met-proinsulin
(SEQ ID NO：49，63)
MWPsp-MWPmp20-(His)
6-EGF-TEV-somatostatin 28
(SEQ ID NO：50，64)
MWPsp-MWPmp20-(His)
6-joint-V8-hyperglycemic-glycogenolytic factor
(SEQ ID NO：51，65)
(2) expression/secretion of fusion product
Expression is by the fused protein of the fusion dna coding of gained among the embodiment 1-7.Fig. 5 is a representative example, is the method that respectively above-mentioned 4 fusion dnas is imported expression vector.
Specifically, with restriction enzyme ApaLI and HindIII (when direction with respect to the M13 primer, when inserting fusion dna with forward) or ApaLI and KpnI (when direction, during) with reverse insertion fusion dna with respect to the M13 primer handle the carrier (pPINS-1, pPINS-2, pSTN, pGCN) that has mixed above-mentioned fusion dna.Then, with restricted fragment through 0.8% agarose gel electrophoresis to isolate dna fragmentation with fusion dna.With dna ligation kit (TakaraShuzo, Co., Ltd.), at 16 ℃, with each an amount of fusion dna of gained thus with used ApaLI and HindIII (when direction in right amount with respect to the M13 primer, when inserting fusion dna with forward) cracked bacillus brevis expression vector pNU211R2L5 (JP-A-5-304962 and JP-A-7-170984) reaction 30 minutes, thus, each fusion dna is imported expression vector.Therefore, expression vector pNU-PINS-1, pNU-PINS-2, pNU-STN and the pNU-GCN of each fusion dna have been mixed respectively.According to currently known methods (Methods in Enzymol., 217:23-33,1993), transform bacillus brevis bacterial strain 47-5Q (FERM BP-1664) with these expression vectors, after this, make the gained transformant at T2 nutrient agar [polypeptone (1%), meat extract (0.5%), yeast extract (0.2%), uridylic (0.1mg/ml), glucose (1%), erythromycin (10 μ g/ml), agar (1.5%), pH7] middle growth.
At 37 ℃, each transformant was cultivated 1 day in T2 substratum (removing agar from the T2 substratum).Then, according to currently known methods (molecular cloning second edition, laboratory manual, cold spring harbor laboratory (1989)), plasmid DNA purification from each substratum, and with ApaLI and HindIII (or KpnI) processing, to determine whether fusion dna has been imported into transformant.For the transformant of having mixed fusion dna, detect expression/secretion by the fused protein of the fusion dna coding that mixes.Specifically, with 1: 1000 volume ratio, the cell suspending liquid that will obtain from the T2 substratum added to 5YC substratum [polypeptone (3%), yeast extract (0.2%), glucose (3%), CaCl respectively
22H
2O (0.01%), MgSO
47H
2O (0.01%), FeSO
47H
2O (0.001%), MnSO
44H
2O (0.001%), ZnSO
47H
2O (0.0001%), glycin (0.3%), erythromycin (10 μ g/ml), pH7] in, 30 ℃ of shaking culture 4 days.
When cultivate finishing,, centrifugal 2 minutes of substratum to obtain supernatant liquor, is used for by currently known methods (Laemmli, U.K., Nature, 227,680-685,1970) electrophoretic analysis protein with 15000rpm.Specifically, 18 each supernatant liquor of μ l are added to 2 μ l damping fluid 1[125mM Tris-HCl (pH6.8), 20% glycerine, 4% SDS, 10% 2 mercapto ethanol] in, boiled 5 minutes, add to 4 μ l damping fluid 2[250mM Tris-HCl (pH6.5) then, 50% glycerine, 0.5% BPB] in.With commercially available 15/25% sds page (Daiichi Chemicals, Co.Ltd., Japan) (electrophoretic buffer is the supernatant liquor of electrophoresis gained: 100mM Tris, 100mM Wheat flavone (Tricine), 0.1% SDS), so that determine whether to exist the expression/secretion of fused protein with Coomassie blue stain.
MWPsp-MWPmp6-, 8-, 9-, 10-, 11-, 12-, 15-, 40-, 50-, 100-(His)
6The insulinogenic expression of-joint-Met-/secretion the results are shown in Fig. 6, as the insulinogenic representative of allogenic polypeptide.Remove MWPsp-MWPmp9-(His)
6Outside-the joint-Met-proinsulin (swimming lane 5), all fusion products all have expression/secretion.MWPsp-proinsulin, MWPsp-MWPmp1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 17-, the insulinogenic expression of 20-, 50-Met-/secretion the results are shown in Fig. 7.Except that MWPsp-proinsulin (swimming lane 3) and MWPsp-MWPmp9-Met-proinsulin (swimming lane 12), all fusion products all have expression/secretion.Also observe higher expression/secretion level for MWPsp-MWPmp6-, 8-, 9-, 10-, 11-, 12-, 15-, 17-, 20-, 50-Met-proinsulin.MWPsp-somatostatin 28, MWPsp-MWPmp10-(His)
6-EGF-TEV-somatostatin 28, MWPsp-MWPmp10-(His)
6-TEV-somatostatin 28 and MWPsp-MWPmp20-(His)
6Expression/the secretion of-EGF-TEV-somatostatin 28 the results are shown in Fig. 8, as the representative of allogenic polypeptide somatostatin 28.Do not observe MWPsp-somatostatin 28 and MWPsp-MWPmp10-(His)
6-TEV-somatostatin 28 has expression/secretion, but observes MWPsp-MWPmp10-(His)
6-EGF-TEV-somatostatin 28 and MWPsp-MWPmp20-(His)
6-EGF-TEV-somatostatin 28 has expression/secretion.And MWPsp-MWPmp20-(His)
6Expression/the secretion level of-EGF-TEV-somatostatin 28 is higher.MWPsp-hyperglycemic-glycogenolytic factor and MWPsp-MWPmp10-, 20-, 30-(His)
6Expression/the secretion of-joint-V8-hyperglycemic-glycogenolytic factor the results are shown in Fig. 9, as the representative of allogenic polypeptide hyperglycemic-glycogenolytic factor.Only observe MWPsp-MWPmp20-(His)
6-joint-V8-hyperglycemic-glycogenolytic factor has expression/secretion.
(3) identify proinsulin
Antibody mediated immunity with Insulinogen C-peptide is identified proinsulin.With 15000rpm with centrifugal 2 minutes of substratum to obtain the supernatant liquor of each substratum.Then, by above-mentioned supernatant liquor electrophoresis, and press currently known methods (Towbin, H. etc., 76,4350-4354,1979) electroblotting to nitrocellulose filter with each 1 microlitre.This film is being dissolved in damping fluid 3[20mM Tris-HCl (pH7.4), 150mM NaCl, 0.1% Tween 20] 5% skimmed milk in soaked 1 hour, then, under oscillating condition, be immersed in again with damping fluid 3 with the rabbit of dilution in 1: 2000 anti--C-peptide antibody (LINCORESEARCH) in, 30 minutes.Under oscillating condition, with damping fluid 3, with described film washing 3 times, each 10 minutes, and then under oscillating condition, be immersed in damping fluid 3 with dilution in 1: 2000 with in the anti-rabbit igg antibody (E-Y laboratory) of peroxidase labelling 30 minutes.After dipping is finished, film is washed 3 times with damping fluid 3 vibrations, each 10 minutes, so that whether determine insulinogenic existence according to the explanation of manufacturers with ECL detection kit (Amersham International plc).Shown in Figure 10 and 11, for MWPsp-MWPmp6-, 8-, 10-, 11-, 12-, 15-, 40-, 50-, 100-(His)
6-joint-Met-proinsulin has detected the signal that the expression proinsulin exists, but for pNU211R2L5 that does not contain fusion dna and MWPsp-MWPmp9-(His)
6-joint-Met-proinsulin does not detect signal.For MWPsp-MWPmp1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 10-, 11-, 12-, 13-, 14-, 15-, 17-, 20-, 50-Met-proinsulin, detected the signal that the expression proinsulin exists, but, do not detect signal for pNU211R2L5, MWPsp-proinsulin and MWPsp-MWPmp9-proinsulin.
(4) insulinogenic cracking
In substratum, cultivate to contain and mixed fusion dna MWPsp-MWPmp10-(His)
6The transformant of-joint-insulinogenic expression vector of Met-.With the substratum of gained centrifugal 15 minutes with 20000rpm.The adding saturation ratio is 30% ammonium sulfate in supernatant liquor.With the supernatant liquor of gained again with centrifugal 20 minutes of 20000rpm to obtain precipitation, the gained resolution of precipitate in an amount of 2mM sodium phosphate buffer (pH7), is dialysed with identical damping fluid.When dialysis finishes, the damping fluid of solution is replaced with 20mM sodium phosphate (pH7) and 150mM NaCl.The solution of gained is added on the chelate column (Pharmacia), and with the same buffer wash-out that contains the 300mM imidazoles so that with fused protein and other impurity protein separation and obtain purifying.Isolated fused protein is also centrifugal as stated above with ammonium sulfate precipitation, collecting precipitation.Solids precipitation is dissolved in the 2mM sodium phosphate buffer (pH7) so that dialyse with identical damping fluid.
Then, formic acid is added in the solution of dialysis, reaches 70% final concentration, to wherein adding cyanogen bromide, the add-on of cyanogen bromide equates with proteinic gram number.Mixture is spent the night in the room temperature placement, so that chemical cracking goes out proinsulin from fused protein.Gained solution is dialysed with 2mM sodium phosphate buffer (pH7), add to then on the chelate column, with the same buffer wash-out proinsulin that contains the 60mM imidazoles.Figure 12 has listed with fusion rotein MWPmp10-(His)
6The result of-joint-Met-proinsulin and proinsulin electrophoresis and Coomassie blue stain on 15/25% polyacrylamide gel, this fusion rotein separates and purifying with chelate column, but also not cracking, described proinsulin has been used bromize fluoride crack.Figure 13 represents to use protein electrophorese, with anti--C-peptide antibody the electrophoresis result trace is identified proinsulin on nitrocellulose filter then.Confirmed insulinogenic existence for fusion rotein.
(5) cracking of somatostatin 28
In substratum, cultivate to contain and mixed fusion dna MWPsp-MWPmp20-(His)
6The transformant of the expression vector of-EGF-TEV-somatostatin 28.With the substratum of gained centrifugal 15 minutes with 20000rpm.The ammonium sulfate that in the gained supernatant liquor, adds 50% saturation ratio.With gained supernatant liquor centrifugal 20 minutes, obtain solids precipitation again, described resolution of precipitate in an amount of 2mM sodium phosphate buffer (pH7), is dialysed with identical damping fluid with 20000rpm.When dialysis finishes, with the damping fluid of 20mM sodium phosphate buffer (pH7) and 150mM NaCl substitutional solution.Gained solution is added on the chelate column (Pharmacia), with the same buffer wash-out that contains the 300mM imidazoles, so that with fused protein MWPmp20-(His)
6-EGF-TEV-somatostatin 28 and other impurity albumen sepn and purifying.(GIBCO BRL 10U) according to the explanation of manufacturers, handles the isolated fused protein (104,52 and 26 microgram) of different amounts, so that cracking goes out somatostatin 28 from fused protein with TEV proteolytic enzyme.Will be with the protein and the untreated protein electrophorese of TEV protease treatment, trace is on nitrocellulose filter, anti-rabbit igg antibody (the E-Y laboratory is diluted 2000 times) with anti-somatostatin antibody of rabbit (MEDAC dilutes 2000 times) and peroxidase labelling detects then.Figure 14 represents to go out somatostatin 28 with TEV albumen enzymatic lysis.
(6) cracking of hyperglycemic-glycogenolytic factor
In substratum, cultivate to contain and mixed fusion dna MWPsp-MWPmp20-(His)
6The transformant of the expression vector of-joint-V8-hyperglycemic-glycogenolytic factor.With the substratum of gained centrifugal 15 minutes with 20000rpm.The ammonium sulfate that in the gained supernatant liquor, adds 50% saturation ratio.With gained supernatant liquor centrifugal 20 minutes, obtain solids precipitation again, described resolution of precipitate in an amount of 2mM sodium phosphate buffer (pH7), is dialysed with identical damping fluid with 20000rpm.When dialysis finishes, with the damping fluid of 20mM sodium phosphate buffer (pH7) and 150mM NaCl substitutional solution.Gained solution is added on the chelate column (Pharmacia), with the same buffer wash-out that contains the 300mM imidazoles, so that with fused protein MWPmp20-(His)
6-joint-V8-hyperglycemic-glycogenolytic factor separates with other impurity and purifying.Handle the different isolated fused proteins of measuring (90,45 and 22.5 microgram) with the 0.1M sodium carbonate solution of V8 proteolytic enzyme (Wako Pure ChemicalIndustries, Ltd., 2 micrograms), so that cracking goes out hyperglycemic-glycogenolytic factor from fused protein.Will be with the protein and the untreated protein electrophorese of V8 protease treatment, trace is on nitrocellulose filter, anti-rabbit igg antibody (the E-Y laboratory is diluted 2000 times) with anti-hyperglycemic-glycogenolytic factor antibody of rabbit (SANBIO dilutes 2000 times) and peroxidase labelling detects then.Figure 14 represents to have gone out hyperglycemic-glycogenolytic factor with V8 albumen enzymatic lysis.
(7) insulinogenic amino acid analysis
Identify from fused protein MWPmp10-(His) by amino acid analysis
6The proinsulin that cracking goes out in-joint-Met-proinsulin.Specifically, analyze and to finish as follows: at Hitachi amino acidanalyser L-8500 (Hitachi, Ltd.) before analyzing on, handle fusion rotein with cyanogen bromide, use then 6N-HCl (containing 0.1% phenol) in 110 ℃ of hydrolysis with the proinsulin of chelate column separation and purification 20 hours.As shown in the table, form basically identical from the insulinogenic amino acid composition and the theoretical amino acid of natural proinsulin of fusion rotein.
Table 1
| Amino acid | Theoretical value | Measured value (nM) | Amino acid is formed |
| A R N+D C | 4 4 4 6 15 11 2 2 12 2 3 3 5 3 4 5 | 3.374 2.977 3.083 1.253 10.634 8.173 1.584 1.212 8.709 1.602 2.304 3.094 2.541 2.211 2.805 4.178 | 4.80 4.24 4.39 1.78 15.13 11.63 2.25 1.72 12.39 2.28 3.28 4.40 3.62 3.15 3.99 5.95 |
| 85 | 59.734 | 85.00 |
(8) assessment output
With fusion product MWPsp-MWPmp10-(His)
6-joint-Met-proinsulin is an example, assesses its oozy output in substratum with the Western trace.To carry out 3 respectively with 1 microlitre supernatant liquor and 1 microlitre proinsulin (Sigma) that 15000rpm obtained in centrifugal 2 minutes
n-doubly dilution, electrophoresis, then trace to nitrocellulose filter so that relatively use the detected strength of signal of anti-C peptide antibody.As shown in figure 16, the strength of signal of the supernatant liquor of 3 times of dilutions insulinogenic similar to 0.03 microgram to 0.1 microgram as if.Therefore, infer and MWPmp10-(His)
6The insulinogenic output of-joint-Met-is 100 to 300mg/ liters.
Structure is mixed with the carrier (pG-GH) of fusion dna MWPsp-MWPmp20-TEV-G-GH
(1) preparation dna fragmentation MWPsp-MWPmp20
According to embodiment 4 (1) described identical methods, the dna fragmentation MWPsp-MWPmp20 of preparation blunt end, different is to repeat 30 circulations: 94 ℃ of sex change 1 minute, 53 ℃ of annealing 1 minute, 72 ℃ of prolongation DNA chains 1 minute; Finish the PCR reaction.
(2) preparation dna fragmentation TEV
According to genetic code table (with above), the chemical synthesis coding aminoacid sequence (AspTyrAspIleProThrThrGluAsnLeuTyrPheGln (SEQ ID NO:2) forward oligonucleotide 5 '-GACTATGATATCCCGACCACTGAAAACCTGTACTTCCAA-3 ' (SEQ ID NO:57) and reverse oligonucleotide 5 '-TTGGAAGTACAGGTTTTCAGTGGTCGGGATATCATAGTC-3 ' (SEQ ID NO:58), this aminoacid sequence can be discerned by TEV proteolytic enzyme.Then, with T4 polynucleotide kinase (Nippon Gene), according to the explanation of manufacturers, with described oligonucleotide phosphorylation, at 95 ℃, 10mM Tris-HCl (pH8) and 5mM MgCl
2Handled 5 minutes in the solution, then 37 ℃ of annealing 15 minutes.TEV handles with phenol with the annealed double chain DNA fragment, uses ethanol sedimentation, and vacuum-drying is dissolved in an amount of distilled water then.
(3) preparation dna fragmentation human growth hormone GH
According to the described identical method of present embodiment (1), the preparation blunt end dna fragmentation GH, different is:
(a) with the plasmid vector that is mixed with dna fragmentation GH as template DNA, prepare this carrier with following method:,, synthesize people's hypophysis cDNA from commercially available people's hypophysis mRNA (Clontech) according to the explanation of manufacturers with the first chain cDNA synthetic agent box (Pharmacia); With Roskam, (nucleic acids research such as W.G., 7,305-320,1979) and Martial, (science such as J.A., 205,602-607,1979) nucleotides sequence of the human growth hormone gene of determining is classified the basis as, synthetic forward primer 5 '-ATGGCTACAGGCTCCCGGAC-3 ' (SEQ ID NO:44) and reverse primer 5 '-CTAGAAGCCACAGCTGCCCT-3 ' (SEQ ID NO:45); Be template and use the synthetic oligonucleotide with above-mentioned cDNA, repeat 35 circulations: handled 1 minute for 94 ℃, 55 ℃ 1 minute, 72 ℃ 1 minute; Finish the PCR reaction; The PCR product that will obtain thus then is that human growth hormone dna is cloned in the pGEM-T carrier (Promega);
(b) forward primer 5 '-TTCCCAACCATTCCCTTATC-3 ' (SEQ ID NO:46) and reverse primer 5 '-CTAGAAGCCACAGCTGCCCT-3 ' (SEQ ID NO:45); With
(c) repeat 25 circulations: 94 ℃ of sex change 1 minute, 55 ℃ of annealing 1 minute, 72 ℃ prolong DNA chain 30 seconds; Finish the PCR reaction.
(4) dna fragmentation of the mutant human growth hormone (G-GH) that is connected with Gly at N-terminal of preparation
According to the described identical method of present embodiment (1), the dna fragmentation G-GH of preparation blunt end, different is: (a) the PCR product GH with the middle gained of 10ng present embodiment (3) is a template DNA; (b) use forward primer 5 '-GGTTTCCCAACCATTCCCTTATC-3 ' (SEQ ID NO:47) and reverse primer 5 '-CTAGAAGCCACAGCTGCCCT-3 ' (SEQ ID NO:45); (c) repeat 25 circulations: 94 ℃ of sex change 1 minute, 55 ℃ of annealing 1 minute, 72 ℃ prolong DNA chain 30 seconds; Finish the PCR reaction.
With T4 polynucleotide kinase (Nippon Gene), by the explanation of manufacturers, the dna fragmentation G-GH of blunt end is carried out phosphorylation reaction, obtain the dna fragmentation G-GH of phosphorylation thus.
(5) preparation fusion dna MWPsp-MWPmp20-TEV
According to the described identical method of present embodiment (1), the fusion dna MWPsp-MWPmp20-TEV of preparation blunt end, different is: (a) at 16 ℃, with dna ligation kit (Takara Shuzo, Co., Ltd.), an amount of dna fragmentation TEV reaction by gained in an amount of dna fragmentation MWPsp-MWPmp20 that will obtain in the present embodiment (1) and the present embodiment (2) 30 minutes prepares the template DNA that is used for PCR reaction; (b) in PCR reaction, use reverse primer 5 '-TTGGAAGTACAGGTTTTC-3 ' (SEQ ID NO:39); (c) repeat 25 circulations: 94 ℃ of sex change 1 minute, 45 ℃ of annealing 1 minute, 72 ℃ with DNA chain extension 30 seconds; Finish the PCR reaction.
After this, with T4 polynucleotide kinase (Nippon Gene), by the explanation of manufacturers, with the PCR product phosphorylation of gained.With dna ligation kit (Takara Shuzo, Co., Ltd), the PCR product of phosphorylation is imported carrier (the BlueScript SK-of HincII cutting, Stratagene) in, so that according to currently known methods (molecular cloning second edition, laboratory manual, cold spring harbor laboratory (1989)) transformed into escherichia coli DH5 α.Plasmid purification carrier DNA from described transformant.In order to confirm to have obtained fusion dna MWPsp-MWPmp20-TEV, the primer forward or backwards (being M13 primer forward or backwards) that is used for definite carrier sequence is determined the nucleotide sequence of described carrier.According to method same as described above, with the carrier that is mixed with MWPsp-MWPmp20-TEV is template DNA, use forward primer 5 '-GTCGTTAACAGTGTATTGCT-3 ' (SEQ ID NO:6) and reverse primer 5 '-TTGGAAGTACAGGTTTTC-3 ' (SEQ ID NO:39) to finish the 2nd PCR reaction, prepare the fusion dna MWPsp-MWPmp20-TEV of blunt end thus.
(6) preparation is mixed with the carrier of fusion dna MWPsp-MWPmp20-TEV-G-GH
According to the described identical method of present embodiment (5), preparation is mixed with the carrier of fusion dna MWPsp-MWPmp20-TEV-G-GH, different is: (a) at 16 ℃, with dna ligation kit (Takara Shuzo, Co., Ltd.), an amount of dna fragmentation G-GH reaction by gained in an amount of fusion dna fragment MWPsp-MWPmp20-TEV that will obtain in the present embodiment (5) and the present embodiment (4) 30 minutes prepares template DNA; (b) with forward primer 5 '-GTCGTTAACAGTGTATTGCT-3 ' (SEQ ID NO:6) and reverse primer 5 '-CTAGAAGCCACAGCTGCCCT-3 ' (SEQ ID NO:45); (c) repeat 25 circulations: 94 ℃ of sex change 1 minute, 53 ℃ of annealing 1 minute, 72 ℃ with DNA chain extension 1 minute; Finish the PCR reaction.
Structure is mixed with the carrier of fusion dna MWPsp-GH
(1) preparation dna fragmentation MWPsp
According to embodiment 1 (1) described identical method, the dna fragmentation MWPsp of preparation blunt end, different is: (a) use reverse primer 5 '-TGCGAAAGCCATTGGAGCAAC-3 ' (SEQ ID NO:34); (b) repeat 30 circulations: 94 ℃ of sex change 1 minute, 53 ℃ of annealing 1 minute, 72 ℃ with DNA chain extension 30 seconds; Finish the PCR reaction.
(2) preparation is mixed with the carrier of fusion dna MWPsp-GH
According to embodiment 9 (5) described identical methods, preparation is mixed with the carrier of fusion dna MWPsp-GH, different is: (a) at 16 ℃, with dna ligation kit (TakaraShuzo, Co., Ltd.), an amount of dna fragmentation GH reaction by gained among an amount of dna fragmentation MWPsp that will obtain in the present embodiment (1) and the embodiment 9 (3) 30 minutes prepares template DNA; (b) with forward primer 5 '-GTCGTTAACAGTGTATTGCT-3 ' (SEQ IDNO:6) and reverse primer 5 '-CTAGAAGCCACAGCTGCCCT-3 ' (SEQ IDNO:45); (c) repeat 25 circulations: 94 ℃ of sex change 1 minute, 53 ℃ of annealing 1 minute, 72 ℃ with DNA chain extension 1 minute; Finish the PCR reaction.
Make up the carrier that is mixed with MWPsp-MWPmp1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 14-, 30-TEV-G-GH respectively
(1) preparation dna fragmentation MWPsp-MWPmp1,2,3,4,5,6,7,8,9,10,11,12,14,30
According to embodiment 9 (1) described identical methods, the preparation blunt end dna fragmentation MWPsp-MWPmp1,2,3,4,5,6,7,8,9,10,11,12,14,30, different is:
(a) with following primer as reverse primer:
MWPmp1：5′-TGCTGCGAAAGCCATTGG-3′(SEQ ID NO：24)
MWPmp2：5′-TTCTGCTGCGAAAGCCAT-3′(SEQ ID NO：25)
MWPmp3：5′-TTCTTCTGCTGCGAAAGC-3′(SEQ ID NO：26)
MWPmp4：5′-TGCTTCTTCTGCTGCGAA-3′(SEQ ID NO：27)
MWPmp5：5′-TGCTGCTTCTTCTGCTGC-3′(SEQ ID NO：28)
MWPmp6：5′-AGTTGCTGCTTCTTCTGC-3′(SEQ ID NO：14)
MWPmp7：5′-AGTAGTTGCTGCTTCTTC-3′(SEQ ID NO：29)
MWPmp8：5′-TGTAGTAGTTGCTGCTTC-3′(SEQ ID NO：15)
MWPmp9：5′-AGCTGTAGTAGTTGCTGC-3′(SEQ ID NO：16)
MWPmp10：5′-TGGAGCTGTAGTAGTTGCTGCTTCTTCTGC-3′
(SEQ ID NO：7)
MWPmp11：5′-TTTTGGAGCTGTAGTAGT-3′(SEQ ID NO：17)
MWPmp12：5′-CATTTTTGGAGCTGTAGT-3′(SEQ ID NO：18)
MWPmp14：5′-AGCGTCCATTTTTGGAGC-3′(SEQ ID NO：31)
MWPmp30：5′-TGCTACCAGGCCAAGAGCTT-3′(SEQ ID NO：43)；
(b) repeat 30 circulations: 94 ℃ of sex change 1 minute, 53 ℃ of annealing 1 minute, 72 ℃ with DNA chain extension 30 seconds; Finish the PCR reaction.
(2) preparation dna fragmentation TEV-G-GH
According to embodiment 9 (1) described identical methods, the dna fragmentation TEV-G-GH of preparation blunt end, different is: (a) with mixing of obtaining among the 10ng embodiment 9 (6) the carrier pG-GH of fusion dna MWPsp-MWPmp20-TEV-G-GH be template DNA; (b) use forward primer 5 '-GACTATGATATCCCGACCACT-3 ' (SEQ ID NO:60) and reverse primer 5 '-CTAGAAGCCACAGCTGCCCT-3 ' (SEQ ID NO:45); (c) repeat 25 circulations: 94 ℃ of sex change 1 minute, 55 ℃ of annealing 1 minute, 72 ℃ prolong DNA chain 30 seconds; Finish the PCR reaction.
With T4 polynucleotide kinase (Nippon Gene),,, obtain the dna fragmentation TEV-G-GH of phosphorylation thus with the dna fragmentation TEV-G-GH phosphorylation of the blunt end of gained by the explanation of manufacturers.
(3) preparation is mixed with the carrier of MWPsp-MWPmp1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 14-, 30-TEV-G-GH respectively
According to embodiment 9 (5) described identical methods, preparation is mixed with MWPsp-MWPmp1-respectively, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 14-, the carrier of 30-TEV-G-GH, different is: (a) at 16 ℃, with dna ligation kit (Takara Shuzo, Co., Ltd.), by an amount of dna fragmentation MWPsp-MWPmp1 that will obtain in the present embodiment (1), 2,3,4,5,6,7,8,9,10,11,12,14,30 with present embodiment (2) in an amount of dna fragmentation TEV-G-GH reaction 30 minutes of gained, prepare template DNA; (b) use forward primer 5 '-GTCGTTAACAGTGTATTGCT-3 ' (SEQ ID NO:6) and reverse primer 5 '-CTAGAAGCCACAGCTGCCCT-3 ' (SEQ ID NO:45); (c) repeat 25 circulations: 94 ℃ of sex change 1 minute, 53 ℃ of annealing 1 minute, 72 ℃ prolong DNA chain 1 minute; Finish the PCR reaction.
Expression/the secretion of fused protein and described product selectivity cracking
(1) nucleotide sequence of the aminoacid sequence of fusion product and the described product of coding
With regard to the fusion product that embodiment 9-11 obtains, the nucleotide sequence of following product and aminoacid sequence list in respectively SEQ ID NOS:52,66 and Figure 17 in.
MWPsp-MWPmp20-TEV-G-GH(SEQ ID NOS：52、66)
(2) expression/secretion of fusion product
Expression is by the fused protein of the fusion dna coding of embodiment 9-11 gained.Figure 18 illustrates, and MWPsp-MWPmp20-TEV-G-GH is imported the method for expression vector.
Specifically, with restriction enzyme ApaLI and HindIII (when direction with respect to the M13 primer that is used to check order, when inserting fusion dna with forward) or ApaLI and KpnI (when direction, during) with reverse insertion fusion dna with respect to the M13 primer that is used to check order handle the carrier that has mixed above-mentioned fusion dna that in embodiment 9-11, makes.Then, with restricted fragment through 0.8% agarose gel electrophoresis to isolate dna fragmentation with fusion dna.With dna ligation kit (Takara Shuzo, Co., Ltd.), at 16 ℃, each an amount of fusion dna of gained thus and an amount of ApaLI and HindIII (or when the direction with respect to the M13 primer, during with reverse insertion fusion dna, use KpnI) the cracked bacillus brevis expression vector pNU211R2L5 (JP-A-5-304962 and JP-A-7-170984) that uses were reacted 30 minutes, thus, with each fusion dna importing expression vector separately.According to currently known methods (Methods in Enzymol., 217:23-33,1993), transform bacillus brevis bacterial strain 47-5Q (FERMBP-1664, JP-A-60-58074 and JP-A-62-201589) with these expression vectors, after this, make the gained transformant at separately T2 nutrient agar [polypeptone (1%), meat extract (0.5%), yeast extract (0.2%), uridylic (0.1mg/ml), glucose (1%), erythromycin (10 μ g/ml), agar (1.5%), pH7] middle growth.
At 37 ℃, respectively transformant was cultivated 1 day in T2 substratum (removing agar from the T2 substratum).Then, according to currently known methods (molecular cloning second edition, laboratory manual, cold spring harbor laboratory (1989)), plasmid DNA purification from each F substratum, and with ApaLI and HindIII (or KpnI) processing, to determine whether fusion dna has been imported into transformant.For the transformant of having mixed fusion dna, detect expression/secretion by the fused protein of the fusion dna coding that mixes.Specifically, with 1: 1000 volume ratio, the cell suspending liquid that will obtain from the T2 substratum added to substratum [polypeptone (3%), yeast extract (0.4%), glucose (3%), MgSO respectively
47H
2O (0.01%), MnSO
44H
2O (0.001%), erythromycin (10 μ g/ml), pH8] in, 30 ℃ of shaking culture 4 days in test tube (2ml/20ml test tube) or Erlenmeyer flask (50ml/500ml).
When cultivate finishing,, centrifugal 2 minutes of substratum to obtain supernatant liquor, is used for by currently known methods (Laemmli, U.K., Nature, 227,680-685,1970) electrophoretic analysis protein with 15000rpm.Specifically, 18 each supernatant liquor of μ l are added to 2 μ l damping fluid 1[125mM Tris-HCl (pH6.8), 20% glycerine, 4% SDS, 10% 2 mercapto ethanol] in, boiled 5 minutes, add to 4 μ l damping fluid 2[250mM Tris-HCl (pH6.5) then, 50% glycerine, 0.5% BPB] in.With commercially available 15/25% sds page (Daiichi Chemicals, Co.Ltd., Japan) (electrophoretic buffer is the supernatant liquor of electrophoresis gained: 100mM Tris, 100mM Wheat flavone (Tricine), 0.1% SDS), so that determine whether to exist the expression/secretion of fused protein with Coomassie blue stain.
Figure 19 has shown following expressing fusion protein/excretory result: the MWP signal peptide MWPsp-GH before human growth hormone just in time wherein; Wherein the MWP signal peptide is just in time at the preceding MWPsp-TEV-G-GH of fusion product TEV-G-GH (being the combination of the recognition sequence and the mutant human growth hormone G-GH of TEV proteolytic enzyme); MWPsp-MWPmp1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 14-, the 30-TEV-G-GH protein of MWP signal peptide wherein by linking to each other with fusion product TEV-G-GH from least one amino-acid residue of MWP protein N end.The electrophorogram of MWPsp-GH is similar to the electrophorogram of the expression product of the carrier pNU211R2L5 that does not contain the allogenic polypeptide gene.Therefore, the MWPsp-GH none is corresponding to the band clearly of tethelin.On the other hand, for MWPsp-TEV-G-GH and MWPsp-MWPmp1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 14-, 30-TEV-G-GH protein, observed Expression of Fusion Protein/secretion (arrow is specified among Figure 19).Compare MWPsp-MWPmp1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 14-, 20-, the higher level of 30-TEV-G-GH protein expression with MWPsp-MWPmp1-TEV-G-GH.
(3) identifier's tethelin GH and mutant human growth hormone G-GH
Personnel selection growth hormone antibody immunity identifier's tethelin and mutant human growth hormone (Western blotting).With the substratum of each transformant of gained in the present embodiment (2) centrifugal 2 minutes, to obtain the supernatant liquor of each substratum with 15000rpm.It is described to press present embodiment (2), with the supernatant liquor electrophoresis of each 1 microlitre, then according to currently known methods (Towbin, H. etc. 76,4350-4354,1979) with its electroblotting to nitrocellulose filter.This film is being dissolved in damping fluid 3[20mM Tris-HCl (pH7.4), 150mM NaCl, 0.1% Tween 20] 5% skimmed milk in soaked 15 minutes, then, under oscillating condition, be immersed in again with damping fluid 3 with 1: 2000 the dilution rabbit anti-HGH antibody (Biostride, Inc.) in 30 minutes.Under oscillating condition, with damping fluid 3, with described film washing 3 times, each 10 minutes, be immersed in then with damping fluid 3 with dilution in 1: 2000 with in the anti-rabbit igg antibody (E-Y laboratory) of peroxidase labelling 30 minutes.After dipping is finished, film with damping fluid 3 washings 3 times, each 10 minutes, is vibrated, so that determine the existence of GH according to the explanation of manufacturers with ECL detection kit (Amersham International plc) simultaneously.As shown in figure 20, all fusion roteins are MWPsp-GH, MWPsp-TEV-G-GH, MWPsp-MWPmp1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 14-, 30-TEV-G-GH, all detected signal, but, do not detect signal for the pNU211R2L5 that does not contain the allogenic polypeptide gene.Just in time be positioned at MWPsp-GH before the human growth hormone for signal peptide, do not detect band corresponding to human growth hormone with Coomassie blue stain and SDS-PAGE, wherein, in SDS-PAGE with Western blotting detection signal.Consider the Western blotting than the Coomassie blue stain sensitivity many, in the time of before the MWP signal peptide is positioned at human growth hormone, MWPsp-GH can express/secrete, but its expression level is low.
(4) cracking of mutant human growth hormone
The transformant overnight incubation that in substratum, will contain the expression vector that has mixed fusion dna MWPsp-MWPmp20-TEV-G-GH.Substratum suspension (volume ratio is 1: 1000) is added in 10 500 milliliters of Erlenmeyer flasks, and each flask respectively contains 50 milliliters of same medium that are used in embodiment 4 (2) expression, cultivates 4 days at 30 ℃.At 4 ℃, with each substratum of gained centrifugal 20 minutes with 10000rpm.Add EDTA, reach the final concentration of 5mM, add ammonium sulfate and reach 60% saturation ratio, to precipitate.Once more with 10000rpm after centrifugal 20 minutes, with solids precipitation be dissolved in an amount of Tris-HCl damping fluid (20mM Tris-HCl, 1mM EDTA, pH8) in, be added to then on Sephadex G-25 (Pharmacia) post, carry out buffer-exchanged.Gained solution is added to and is adsorbed onto used buffer A [20mMTris-HCl, 1mM EDTA, 1M urea, 20% propyl alcohol, pH8] (Pharmacia QXL) on the post, uses buffer B (buffer A+1M NaCl) to carry out gradient elution to the anionite-exchange resin crossed of balance again.Anti-HGH's antibody positive fraction that will elute with 220-300mM NaCl is with Ultrafree (Millipore Corp., UFV2BCC40) concentrate, use damping fluid C (0.1%TFA, 10% acetonitrile) to replace simultaneously, be added to then on the RPC post (Pharmacia), carry out reverse-phase chromatography.Use damping fluid D[0.1% TFA subsequently, 60% acetonitrile] result that carries out gradient elution is, target fusion rotein MWPmp20-TEV-G-GH by the acetonitrile wash-out of 45-50% get off.The fusion rotein that obtains is thus dialysed with 2mM Tris-HCl (pH8), use the TEV protease treatment then.According to the explanation of manufacturers, (GIBCO BRL 5U) handles 5 microgram fusion roteins, goes out mutant human growth hormone G-GH with cracking with TEV proteolytic enzyme.Figure 21 and 22 is SDS-PAGE and the Western trace figure that represent the cracking result respectively.According to finishing SDS-PAGE and Western trace with present embodiment (2) and (3) described identical method.With regard to Figure 21 and 22, on the position (arrow indicate) identical with commercially available human growth hormone (positive control), cracking has gone out the mutant human growth hormone G-GH that has extra Gly at N-terminal.
Also can express other polypeptide hNGF, mLIF, bSCF and hPDGF-B according to the method identical with present embodiment.When the amino acid number from the MWP N-terminal is 10,40 or 100, do not observe secretion.This shows the type that depends on used allogenic polypeptide by the secretory chance that causes with at least one amino acid whose fusion from the MWP N-terminal.
By with the new fusion of exogenous protein, the present invention can carry out high-caliber expression/secretion, also can produce natural protein by chemistry or enzymatic selective splitting.
All documents that this paper quoted comprise that patent application all is incorporated herein by reference in full.
Following is SEQ ID NOS:48-52 as herein described, the sequence data of 62-66: SEQ ID NO:48:
gtcgttaaca gtgtattggc tagtgcactc gcacttactg ttgctccaat ggctttcgca 60
gcagaagaag cagcaactac tacagctcca catcatcatc atcatcacgg ttctccagta 120
ccttctggaa tgtttgtgaa ccaacacctg tgcggctcac acctggtgga agctctctac 180
ctagtgtgcg gggaaagagg cttcttctac acacccaaga cccgccggga ggcagaggac 240
ctgcaggtgg ggcaggtgga gctgggcggg ggccctggtg caggcagcct gcagcccttg 300
gccctggagg ggtccctgca gaagcgtggc attgtggaac aatgctgtac cagcatctgc 360
tccctctacc agctggagaa ctactgcaac 390
SEQ ID NO：49：
gtcgttaaca gtgtattggc tagtgcactc gcacttactg ttgctccaat ggctttcgca 60
gcagaagaag cagcaactac tacagctcca atgtttgtga accaacacct gtgcggctca 120
cacctggtgg aagctctcta cctagtgtgc ggggaaagag gcttcttcta cacacccaag 180
acccgccggg aggcagagga cctgcaggtg gggcaggtgg agctgggcgg gggccctggt 240
gcaggcagcc tgcagccctt ggccctggag gggtccctgc agaagcgtgg cattgtggaa 300
caatgctgta ccagcatctg ctccctctac cagctggaga actactgcaa c 351
SEQ ID NO：50：
gtcgttaaca gtgtattggc tagtgcactc gcacttactg ttgctccaat ggctttcgca 60
gcagaagaag cagcaactac tacagctcca aaaatggacg ctgatatgga aaaaaccgta 120
catcatcatc atcatcacaa ctctgactcc gaatgcccgc tgtctcacga cggttattgc 180
ctgcatgatg gtgtttgtat gtatatcgaa gctctggaca aatatgcttg caactgtgtt 240
gttggttaca tcggtgagcg ttgccagtat cgcgacctga aatggtggga actgcgtgac 300
tatgatatcc cgaccactga aaacctgtac ttccaatctg ctaactcaaa cccggctatg 360
gcaccccgag aacgcaaagc tggctgcaag aatttcttct ggaagacttt cacatcctgt 420
SEQ ID NO：51：
gtcgttaaca gtgtattggc tagtgcactc gcacttactg ttgctccaat ggctttcgca 60
gcagaagaag cagcaactac tacagctcca aaaatggacg ctgatatgga aaaaaccgta 120
catcatcatc atcatcacgg ttctccagta ccttctggat tcctggaaca cagccaaggt 180
actttcacat ccgactactc taaatatctg gattcccgtc gcgctcaaga tttcgttcaa 240
tggctgatga acact 255
SEQ ID NO：52：
gtcgttaaca gtgtattggc tagtgcactc gcacttactg ttgctccaat ggctttcgca 60
gcagaagaag cagcaactac tacagctcca aaaatggacg ctgatatgga aaaaaccgta 120
gactatgata tcccgaccac tgaaaacctg tacttccaag gtttcccaac cattccctta 180
tccaggcttt ttgacaacgc tatgctccgc gcccatcgtc tgcaccagct ggcctttgac 240
acctaccagg agtttgaaga agcctatatc ccaaaggaac agaagtattc attcctgcag 300
aacccccaga cctccctctg tttctcagag tctattccga caccctccaa cagggaggaa 360
acacaacaga aatccaacct agagctgctc cgcatctccc tgctgctcat ccagtcgtgg 420
ctggagcccg tgcagttcct caggagtgtc ttcgccaaca gcctggtgta cggcgcctct 480
gacagcaacg tctatgacct cctaaaggac ctagaggaag gcatccaaac gctgatgggg 540
aggctggaag atggcagccc ccggactggg cagatcttca agcagaccta cagcaagttc 600
gacacaaact cacacaacga tgacgcacta ctcaagaact acgggctgct ctactgcttc 660
aggaaggaca tggacaaggt cgagacattc ctgcgcatcg tgcagtgccg ctctgtggag 720
ggcagctgtg gcttc 735
SEQ NO ID：62：
Val Val Asn Ser Val Leu Ala Ser Ala Leu Ala Leu Thr Val Ala Pro
1 5 10 15
Met Ala Phe Ala Ala Glu Glu Ala Ala Thr Thr Thr Ala Pro His His
20 25 30
His His His His Gly Ser Pro Val Pro Ser Gly Met Phe Val Asn Gln
35 40 45
His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly
50 55 60
Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp
65 70 75 80
Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser
85 90 95
Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val
100 105 110
Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr
115 120 125
Cys Asn
130
SEQ ID NO：63：
Val Val Asn Ser Val Leu Ala Ser Ala Leu Ala Leu Thr Val Ala Pro
1 5 10 15
Met Ala Phe Ala Ala Glu Glu Ala Ala Thr Thr Thr Ala Pro Met Phe
20 25 30
Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu
35 40 45
Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu
50 55 60
Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly
65 70 75 80
Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg
85 90 95
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu
100 105 110
Glu Asn Tyr Cys Asn
115
SEQ ID NO：64：
Val Val Asn Ser Val Leu Ala Ser Ala Leu Ala Leu Thr Val Ala Pro
1 5 10 15
Met Ala Phe Ala Ala Glu Glu Ala Ala Thr Thr Thr Ala Pro Lys Met
20 25 30
Asp Ala Asp Met Glu Lys Thr Val His His His His His His Asn Ser
35 40 45
Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly
50 55 60
Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val
65 70 75 80
Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp
85 90 95
Glu Leu Arg Asp Tyr Asp Ile Pro Thr Thr Glu Asn Leu Tyr Phe Gln
100 105 110
Ser Ala Asn Ser Asn Pro Ala Met Ala Pro Arg Glu Arg Lys Ala Gly
115 120 125
Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys
130 135 140
SEQ ID NO：65：
Val Val Asn Ser Val Leu Ala Ser Ala Leu Ala Leu Thr Val Ala Pro
1 5 10 15
Met Ala Phe Ala Ala Glu Glu Ala Ala Thr Thr Thr Ala Pro Lys Met
20 25 30
Asp Ala Asp Met Glu Lys Thr Val His His His His His His Gly Ser
35 40 45
Pro Val Pro Ser Gly Phe Leu Glu His Ser Gln Gly Thr Phe Thr Ser
50 55 60
Asp Tyr Ser Lys Tyr Leu Asp Ser Arg Arg Ala Gln Asp Phe Val Gln
65 70 75 80
Trp Leu Met Asn Thr
85
SEQ ID NO：66：
Val Val Asn Ser Val Leu Ala Ser Ala Leu Ala Leu Thr Val Ala Pro
1 5 10 15
Met Ala Phe Ala Ala Glu Glu Ala Ala Thr Thr Thr Ala Pro Lys Met
20 25 30
Asp Ala Asp Met Glu Lys Thr Val Asp Tyr Asp Ile Pro Thr Thr Glu
35 40 45
Asn Leu Tyr Phe Gln Gly Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe
50 55 60
Asp Asn Ala Met Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp
65 70 75 80
Thr Tyr Gln Glu Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr
85 90 95
Ser Phe Leu Gln Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile
100 105 110
Pro Thr Pro Ser Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu
115 120 125
Leu Leu Arg Ile Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val
130 135 140
Gln Phe Leu Arg Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser
145 150 155 160
Asp Ser Asn Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln
165 170 175
Thr Leu Met Gly Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile
180 185 190
Phe Lys Gln Thr Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp
195 200 205
Ala Leu Leu Lys Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met
210 215 220
Asp Lys Val Glu Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu
225 230 235 240
Gly Ser Cys Gly Phe
245
Claims (20)
1. separated DNA, it can produce allogenic polypeptide in bacillus brevis (Bacillus brevis), comprise being connected and encode and comprise first of linearity connection with 3 ' end of the nucleotide sequence in bacillus promoter district, the nucleotide sequence of the fusion rotein of the second and the 3rd sequence, the sequence that consists of n the amino-acid residue that begins from bacillus cell wall-held protein (CWP) N-terminal of wherein said first sequence, wherein when exogenous peptide behaviour proinsulin, n is 6,7,8,10,11,12,15,17,20 or 50, when exogenous peptide was Porcine glucagon, n was 20, and when allogenic polypeptide is mutant human tethelin, n is 1-12,14,20 or 30, one or more amino-acid residues that consist of of second sequence are used for by chemistry or enzyme process described the 3rd sequence and described first sequence being cut, described second sequence does not exist in the 3rd sequence, and the allogenic polypeptide sequence that the 3rd sequence comprises is selected from people's proinsulin, Porcine glucagon, with the mutant human tethelin that has glycine or Serine at N-terminal, described thus the 3rd sequence can only be cut at described second sequence place and described first sequence by chemistry or enzyme process.
2. the described DNA of claim 1, wherein said fusion rotein also comprises the signal peptide sequence of bacillus cell wall-held protein at its N-terminal.
3. claim 1 or 2 described DNA, wherein said fusion rotein also comprise by as the sequence of separating and the amino-acid residue of the mark that purifying is used is formed.
4. the DNA of claim 3, the wherein said His6 that is labeled as.
5. claim 1 or 2 described DNA, wherein said fusion rotein also comprise the sequence of forming by as the amino-acid residue of joint.
6. claim 1 or 2 described DNA, wherein said genus bacillus is a bacillus brevis.
7. claim 1 or 2 described DNA, wherein described second sequence of described the 3rd sequence and described first sequence being cut by chemical method is a methionine(Met).
8. claim 1 or 2 described DNA, wherein comprise can be by albumen enzymatic cracked sequence for described second sequence of described the 3rd sequence and described first sequence being cut by enzyme process.
9. the described DNA of claim 1, wherein said first sequence consist of 6,7,8,10,11,12,15,17 of the proteic N-terminal of cell wall protein midfeather, 20 or 50 amino-acid residues; Described second sequence comprises by the methionine residues of chemical method with described the 3rd sequence and the incision of described first sequence; And described the 3rd sequence does not contain methionine(Met) in its aminoacid sequence and be people's proinsulin; And described fusion rotein further comprises the 4th sequence of being made up of 6 histidine residues as separation and purifying mark, and comprise and contain five sequence of SEQ ID NO:1 as the aminoacid sequence of joint, the described the 4th and the 5th sequence described first and described second sequence between linear according to the order of sequence each other the connection.
10. the described DNA of claim 9, wherein said fusion rotein also comprises midfeather protein signal peptide sequence at its N-terminal.
11. the DNA of claim 1,20 amino-acid residues that consist of cell wall protein midfeather protein N terminal of wherein said first sequence; Described second sequence comprises Phe Leu Glu aminoacid sequence, is used for by V8 proteolytic enzyme enzyme process described the 3rd sequence and described first sequence being cut; And the peptide sequence that comprises of described the 3rd sequence does not contain L-glutamic acid in its aminoacid sequence and be Porcine glucagon; And described fusion rotein further comprises and contains six histidine residues as separating and the 4th sequence of purifying usefulness mark, and comprises five sequence of SEQ ID NO:1 aminoacid sequence as joint; The the described the 4th and the 5th sequence described first and described second sequence between linear according to the order of sequence each other the connection.
12. the described DNA of claim 11, wherein said fusion rotein also comprises midfeather protein signal peptide sequence at its N-terminal.
13. the described DNA of claim 1, wherein said first sequence is by the 1-12 of the proteic N-end of cell wall protein midfeather, and 14,20 or 30 amino-acid residues are formed; Described second sequence comprises the aminoacid sequence of SEQ ID NO:2, is used for by TEV proteolytic enzyme enzyme described the 3rd sequence and described first sequence being cut; And described the 3rd sequence is included in and does not contain the TEV protease recognition sequence in its aminoacid sequence and be the peptide sequence that N-terminal has the mutant human tethelin of glycine or Serine.
14. the described DNA of claim 13, wherein said fusion rotein also comprises midfeather protein signal peptide sequence at its N-terminal.
15. separated DNA, it can produce human somatotropin's statin 28 polypeptide in bacillus brevis, the nucleotide sequence that comprises encoding fusion protein, wherein fusion rotein comprises first of linear connection according to the order of sequence, second, third and fourth sequence, wherein 10 or 20 amino-acid residues that consist of genus bacillus midfeather protein N terminal of first sequence; Second sequence comprises that the human epidermal growth factor is as joint; The 3rd sequence comprises SEQ ID NO:2 aminoacid sequence, is used for by TEV proteolytic enzyme enzyme process the 4th sequence and first sequence being cut; And the 4th sequence human somatotropin statin 28 polypeptide do not contain the TEV protease recognition sequence and have glycine or Serine at N-terminal in its aminoacid sequence, and described fusion rotein further comprises and consists of six histidine residues as separating and the 5th sequence of purifying usefulness mark, described the 5th sequence is linear according to the order of sequence the connection between described first and second sequences, and wherein said Nucleotide is connected to 3 ' end of the nucleotide sequence that contains the bacillus promoter district.
16. the described DNA of claim 15, wherein said fusion rotein also comprises midfeather protein signal peptide sequence at its N-terminal.
17. the DNA of claim 15 or 16, wherein said genus bacillus is a bacillus brevis.
18. comprise carrier according to each described DNA in the claim 1 to 17.
19. bacillus brevis by the described carrier conversion of claim 18.
20. a method for preparing recombinant polypeptide comprises: in substratum, cultivate the described bacterium of claim 19, the fusion rotein that comprises allogenic polypeptide is gathered with extracellular on this bacterium; From substratum, isolate fusion rotein; From the isolated fusion rotein of institute, described allogenic polypeptide cracking is come out; With the described polypeptide of collection.
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| WO1993017098A1 (en) * | 1992-02-19 | 1993-09-02 | The State Of Oregon Acting By And Through The Oregon State Board Of Higher Education On Behalf Of Oregon State University | Production of viral resistant plants via introduction of untranslatable plus sense viral rna |
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| EP0409113A1 (en) * | 1989-07-20 | 1991-01-23 | BEHRINGWERKE Aktiengesellschaft | Human erythropoietin muteins, their production and their use |
| WO1993017098A1 (en) * | 1992-02-19 | 1993-09-02 | The State Of Oregon Acting By And Through The Oregon State Board Of Higher Education On Behalf Of Oregon State University | Production of viral resistant plants via introduction of untranslatable plus sense viral rna |
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| CN1289780A (en) | 2001-04-04 |
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