CN1258744A - Polypeptide ligase being stable in non-water medium and its preparation and application - Google Patents
Polypeptide ligase being stable in non-water medium and its preparation and application Download PDFInfo
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Abstract
本发明提供了一种衍生自枯草蛋白酶E的变体酶,它含有Ser221Cys、Met222Ala和Pro225Ala突变。较佳地,它还含有选自下组的突变:Asn118Ser和Ser24His/Lys27Asp/Ser236CYs。本发明的变体酶在非水介质中的稳定性及热稳定性都显著提高,因而可用于非水介质中的多肽连接反应。本发明还提供了相应的编码序列、表达载体、转化的宿主细胞和制备方法。The present invention provides a variant enzyme derived from subtilisin E, which contains Ser221Cys, Met222Ala and Pro225Ala mutations. Preferably, it also contains mutations selected from the group consisting of: Asn118Ser and Ser24His/Lys27Asp/Ser236CYs. The stability and thermostability of the mutant enzyme in the non-aqueous medium are significantly improved, so it can be used in the polypeptide linking reaction in the non-aqueous medium. The invention also provides corresponding coding sequence, expression vector, transformed host cell and preparation method.
Description
The present invention relates to genetically engineered and enzyme engineering field, more specifically, relate to the new polypeptide ligase enzyme that is applicable to non-aqueous media.
Studies show that the active centre Ser221 of subtilisin BPN ' just becomes the polypeptide ligase enzyme by lytic enzyme after being replaced by Cys.In these polypeptide ligase enzymes, Subtiligase is present best variant enzyme, and has been applied to synthetic (Jackson:J.Am.Chem.Soc.1995,117,819-20 and Science 1994,266, the 243-7 of RNase A enzyme and cyclic peptide; Chang:Proc.Natl.Acad.Sci.USA 1994,91,12544-8).This mutant of Subtiligase is that (Abrahmsen:WO 94/18329 through the Ser221Cys/Pro225Ala pair of polypeptide ligase enzyme that is used for the aqueous solution that suddenlys change and get for BPN '; U.S. Patent No. 5,403,737; Biochemistry 1991,30,4151-9).In addition, also obtained some other mutant on this (referring to Subtiligase) basis, but all do not mentioned the stability of these mutant, especially the stability in non-aqueous media is improved.
Yet, except in water, carry out polypeptide synthetic, it is synthetic usually also need to carry out polypeptide in non-aqueous media (especially organic solvent).Therefore, this area presses for the polypeptide ligase enzyme that can be applicable to non-aqueous media always.
Purpose of the present invention is exactly the above-mentioned shortcoming that overcomes in the prior art, provide a kind of in non-aqueous media stability high, have polypeptide chain and connect active enzyme, thereby solve the problem of the poor stability of polypeptide ligase enzyme in non-aqueous media, and help improving the solvability that chemically modified-enzyme connects substrate in the technology.
In one aspect of the invention, provide a kind of polypeptide ligase enzyme, it is the variant enzyme of subtilisin E, it is characterized in that, it contains Ser221Cys, Met222Ala and Pro225Ala sudden change.Preferably, this polypeptide ligase enzyme also contains the sudden change that is selected from down group: Asn118Ser, and Ser24His/Lys27Asp/Ser236Cys.
In another aspect of this invention, provide a kind of separated DNA sequence, its a kind of polypeptide ligase enzyme of encoding, this ligase enzyme is the variant enzyme of subtilisin E, and it contains Ser221Cys, Met222Ala and Pro225Ala sudden change.
In another aspect of this invention, provide a kind of expression vector, it contains the dna sequence dna of above-mentioned code book invention polypeptide ligase enzyme.In addition, also provide a kind of host cell, this host cell is transformed by above-mentioned expression vector.
In another aspect of this invention, provide a kind of method of producing novel polypeptide ligase enzyme of the present invention, the method comprising the steps of:
(a) will the encode dna sequence dna of this polypeptide ligase enzyme operationally is connected in expression regulation sequence, forms polypeptide and connects the expression of enzymes carrier;
(b) expression vector in the step (a) is transformed into host cell;
(c) under the condition that is fit to this polypeptide ligase enzyme of expression, cultivate, thereby give expression to this polypeptide ligase enzyme;
(d) separation and purification goes out this polypeptide ligase enzyme.
On the other hand, provide the purposes of polypeptide ligase enzyme of the present invention, it is used to the polypeptide ligation in the non-aqueous media.
In addition, the present invention also provides the novel process of separation and purification polypeptide ligase enzyme of the present invention, it comprises: the supernatant 30%-60% ammonium sulfate precipitation that will ferment, throw out dissolves after DEAE-Dextran A-25 post and Acrylex P-150 post with the phosphoric acid buffer of pH6.0-7.4.
In appended accompanying drawing,
Fig. 1 be a kind of variant enzyme BHG of the present invention at 60 ℃, the beta stability line figure among the 60%DMF;
Fig. 2 is under the DMF of different concns, variant enzyme BHG activity curve figure;
Fig. 3 is under the DMF of different concns, the another kind of variant enzyme BW of the present invention activity curve figure.
The present invention is based on and finishes on the following unexpected basis of finding: the 222nd methionine (Met) among withered grass (bacillus) the protease E is sported alanine (Ala) afterwards, can significantly improve the stability of albumen in non-aqueous media. In conjunction with Ser221Cys and Pro225Ala (these two sudden changes become the polypeptide ligase with subtilopeptidase A by hydrolase), just formed of the present invention in non-aqueous media high and the ligase with taking over a job property of polypeptide chain of stability.
In the present invention, " subtilisin E " refers to the protease E from hay bacillus, and it can be the protease E of natural wild type, also can be the protease E that biology is derived or recombinated. The amino acid sequence of the subtilisin E of natural wild type is shown among the SEQ ID No.3.
As used herein, term " non-aqueous media " refers to contain the medium of organic solvent, and this medium can be only to contain a kind of organic solvent, also can be the mixed organic solvents that two or more organic solvents form. Can be the mixed solvent of the formation of organic solvent and water in addition. Representativeness is that organic solvent has: acetonitrile, methyl-sulfoxide, dimethyl formamide (DMF) etc. Preferably, contain DMF in this non-aqueous media.
New polypeptide ligase of the present invention can be prepared like this: the sequence according to published subtilisin E is come synthetic primer, amplifies the coded sequence of subtilisin E from hay bacillus by the PCR method. Then, with the dna sequence dna of subtilisin E, carry out genetic modification by the sudden change form of pointing out among the present invention, the technology of carrying out genetic modification is as known in the art, for example can referring to "Mutagenesis： a Practical Approach”, M.J.McPherson, Ed., (UK. (1991) wherein for example comprises direct mutagenesis, cassette mutagenesis and polymerase chain reaction (PCR) mutagenesis for IRL Press, Oxford. Having obtained it to be connected into suitable expression vector after the code book behind the rite-directed mutagenesis invents the dna sequence dna of new polypeptide ligase, changing suitable host cell over to. At last, cultivate the host cell after transforming, obtain new polypeptide ligase of the present invention by separation and purification.
In the present invention, can select various carrier known in the art, such as commercially available carrier. Such as, select commercially available carrier, the nucleotide sequence of then code book being invented new polypeptide ligase operationally is connected in expression regulation sequence, can form protein expression vector.
As used herein, " operationally being connected in " refers to so a kind of situation, and namely some part of linear DNA sequence can affect the activity of same other parts of linear DNA sequence. For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion targeting sequencing) DNA operationally is connected in polypeptid DNA so; If transcribing of promoter control sequence, it is operationally to be connected in coded sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in coded sequence so. Generally, " operationally being connected in " means adjoining, then means in reading frame adjacent for the secretion targeting sequencing.
In the present invention, term " host cell " comprises prokaryotic and eukaryotic. The example of protokaryon host cell commonly used comprises Escherichia coli, hay bacillus etc. Eukaryotic host cell commonly used comprises yeast cells, insect cell and mammalian cell. Preferably, this host cell is prokaryotic, more preferably is hay bacillus.
The inventor through for many years research, with 118Ser, 222Ala, 60Asn, 218Ser, the various sudden change forms such as 103Arg and combination thereof are introduced in subtilisin E and the two mutant of 221Cys/225Ala, and in conjunction with the stability of computer research mutain under the non-aqueous media environment. Found that, the 222nd methionine (Met) among withered grass (bacillus) the protease E is sported alanine (Ala) afterwards (as forming Ser221Cys/Met222Ala/Pro225Ala three mutant), can significantly improve the stability of albumen in non-aqueous media. In addition, by the further mutagenesis on other sites, can be created in the higher and/or active more much higher peptide ligase of stability in the non-aqueous media.
In one embodiment of the invention, also 118 are suddenlyd change, thereby formed Asn118Ser/Ser221Cys/Met222Ala/Pro225Ala four mutant, this mutant enzyme (referred to as variant enzyme) is named as BHG.
In another embodiment of the present invention, also 24,27 and 236 are suddenlyd change, thereby formed Ser24His/Lys27Asp/Ser221Cys/Met222Ala/Pro225Ala/Ser236Cy s six mutant, this mutant enzyme (referred to as variant enzyme) is named as BW.
Mutant polypeptide ligase of the present invention has stable superiority at energy, thereby not only in water, and in non-aqueous media, also have very high stability, be highly suitable for the polypeptide coupled reaction (for example polypeptide synthetic reaction) of in containing the system of organic solvent, carrying out.
In the art, it has been generally acknowledged that subtilisin E through the variant enzyme after the Ser221Cys sudden change can not be ripe during at secreting, expressing (Abrahmsen:Biochemistry 1991,30,4151-9), the bacterial classification coexpression that therefore must add wild-type during the fermentation is to help the maturation of variant enzyme.Yet this is extremely unfavorable to purifying work, especially because the nature difference between wild-type enzyme and the variant enzyme is very little, so be difficult to separate; Simultaneously, wild-type enzyme is a kind of proteolytic enzyme, has extremely strong hydrolytic activity, so meeting hydrolysis variant enzyme, and this is also unfavorable to variant enzyme.
Yet the inventor is unexpected to be found, new polypeptide ligase enzyme of the present invention (for example Asn118Ser/Ser221Cys/Met222Ala/Pro225Ala variant enzyme BHG) can be ripe voluntarily in expression, purge process, need not the help of wild-type enzyme.Therefore, the separation and purification program be can simplify greatly, and purification efficiency and purity improved greatly.In one embodiment, the host cell after the conversion of the present invention is in 2YT, and 37 ℃ fermented 30-36 hour, and found that ripe variant enzyme BHG accounted for 36% of total protein.To fermentation supernatant 30%-60% ammonium sulfate precipitation, throw out dissolves after DEAE-Dextran A-25 post and Acrylex P-150 post obtain target protein with the phosphoric acid buffer of pH6.2, and purity can reach electrophoresis pure (more than 95%).
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The plasmid construction of BHG and BW variant enzyme and expression
The EcoR I/Pst I site that the gene of subtilisin E (comprise signal peptide and lead the gene of peptide moiety, altogether 1.9Kb) is inserted pAlter-I (purchasing the company in Promega).When rite-directed mutagenesis, suddenly change with reference to Promega company sudden change specification sheets, wherein, used mutagenic primer is:
The Ser221Cys/Met222Ala/Pro225Ala primer, 5 '-AAC GGA ACG T
GC
GCG GCGACT GCT CAC GTT GCC;
The Asn118Ser primer, 5 '-ATT
TCC AAC
TCT ATG GAT GTT;
The Ser236Cys primer, 5 '-GCG TTA ATT CTT T
GCAAG CAC CCG;
The Ser24His/Lys27Asp primer, 5 '-CAC AG GC
C ATA ACG TA
GA
CG TAG CTGT;
In above-mentioned primer sequence, tiltedly the mutational site represented in boldface type, and " Ser221Cys " expression becomes Cys with the 221st Ser in the original acid sequence, and all the other mutational sites are represented with same way as.
Cut out 1.9Kb fragment after the sudden change with EcoR I/Pst I, insert (Guo Xinghua: biotechnology journal 1991 in the pBE-2 shuttle plasmid, 7 (3), 224-9), obtain expression plasmid pBHG and pBW, they express variant enzyme BHG (Asn118Ser/Ser221Cys/Met222Ala/Pro225Ala four mutant) and BW (Ser24His/Lys27Asp/Ser221Cys/Met222Ala/Pro225Ala/Ser236Cy s) respectively in Bacillus subtilus DB104.
Wherein, the Bacillus subtilus BHG (AprE) that expresses variant enzyme BHG is preserved in Chinese typical culture collection center C CTCC (China, Wuhan) on December 11st, 1998, and preserving number is CCTCC M98022.
In addition, the dna encoding sequence of variant enzyme BHG and the aminoacid sequence after the maturation are shown in respectively among SEQ IDNo.1 and the SEQ ID No.2.
Embodiment 2
The separation and purification of variant enzyme
For the 2L fermentation supernatant of engineering bacteria BHG, with (NH
4)
2SO
4After the precipitation (30% to 60% saturation ratio), be dissolved in 15ml 10mM phosphoric acid buffer (1mM CaCl
21mM EDTA, pH6.5), directly go up the DEAE-DextranA-25 post, with slowly drip washing (20ml/hr.) of phosphoric acid buffer (pH6.5), SDS-PAGE identifies desirable proteins, and super worry concentrates goes up the AcrylexP-150 post, with slowly drip washing of phosphoric acid buffer (pH6.5), obtain the variant enzyme BHG of electrophoresis pure (95%).
Embodiment 3
Performance measurement
(a) dynamic analysis:
Press Abrahmsen:Biochemistry (1991), 30, the method described in the 4151-9 is measured esterlysis activity and amido bond hydrolytic activity.When the esterlysis of mensuration variant enzyme BHG in DMF was active, DMF accounted for 10%, 20%, 40% and 60% respectively in the system.
(b) stability of variant enzyme:
Variant enzyme is placed 60%DMF, in 60 ℃ of insulations, the residue esterlysis activity of per 1 hour test sample product.
In addition, under similarity condition, measure esterlysis activity and the amidohydrolase activity of Subtiligase, in contrast.
Test data is listed in table 1 and table 2, the result shows: the amidohydrolase activity of variant enzyme is higher 4.5 times than Subtiligase, esterlysis is active high 1.5 times, though the active Subtiligase frequently with amidohydrolase activity of the esterlysis of variant enzyme hangs down 3 times, but in the mixed system of DMF-water, DMF is little to variant enzyme BHG activity influence, the activity of variant enzyme only descend about 6 times (Fig. 2) in the 60%DMF system; And Subtiligase is very unstable in DMF, and the activity in the 5%DMF system descends about 70 times.
Fig. 1 shows that also variant enzyme BHG is at 60 ℃, and the half life among the 60%DMF reaches 17 hours.More than this variant enzyme of explanation not only has stability preferably in DMF, and thermostability is preferably arranged.Studies show that, the carbonyl of the hydroxyl hydrogen of 118Ser and solvent DMF forms hydrogen bond in BHG, ketonic oxygen on the 119Met main chain is by a part water and solvent DMF effect, hydrogen of the imino-on the side chain of 27Lys and solvent DMF form hydrogen bond, main chain is tended towards stability, remedied from water and transformed the hydrogen bond that is lost, thereby in DMF, shown stronger stability to organic phase.
Fig. 3 shows that BW also has stronger stability in DMF, and the activity of BW increases with the rising of solvent strength.
In sum.New polypeptide ligase enzyme of the present invention, as variant enzyme BHG by the aqueous solution when non-aqueous system transforms, the active reduction, but solvent is little to active influence.Still having advantages of higher stability under 60%DMF, 60 ℃ of conditions, is polypeptide ligase enzyme stable in the non-aqueous media therefore.This also is the report of present relevant first polypeptide ligase enzyme stability in non-aqueous media.
Table 1 BHG and the BW esterlysis kinetic constant in DMF
| Enzyme | Solvent (DMF) | Kcat (s-1) | ???Km ???(M) | ?Kcat/Km ?(M-1s-1) |
| ? | 0％ | 2.88 | ?8.94×10 -6 | ?3.22×10 5 |
| 10％ | 84.70 | ?6.4×10 -4 | ?1.32×10 5 | |
| 20％ | 10.92 | ?7.79×10 -5 | ?1.40×10 5 | |
| 40％ | 20.80 | ?1.18×10 -4 | ?1.76×10 5 | |
| 60％ | 52.44 | ?9.29×10 -4 | ?5.64×10 4 | |
| ?Subtiligase | 0％ | 4.1×10 | ?1.9×10 -4 | ?2.1×10 5 |
| 5％ | 11.38 | ?3.76×10 -3 | ?3.03×10 3 | |
| ? | 0％ | 6.19 | ?6.76×10 -5 | ?9.16×10 4 |
| 10％ | 13.89 | ?8.25×10 -5 | ?1.68×10 5 | |
| 20％ | 16.36 | ?6.05×10 -5 | ?2.70×10 5 | |
| 40％ | 40.05 | ?8.56×10 -5 | ?4.68×10 5 |
The hydrolysis acid amides of table 2 BHG and the kinetic constant of esterlysis
| Enzyme | ????????s-Ala-ala-Pro-Phe-pNA | ?????s-Ala-Ala-Pro-Phe-Sbz | Esterase/Ntn hydrolase | ||||
| Kcat (s -1) | Km (M) | Kcat/Km (M -1s -1) | Kcat (s -1) | Km (M) | Kcat/Km (M -1s -1) | ||
| Subtiligase | 4.53×10 -5 | 2.74×10 -4 | 0.1653 | 4.1×10 | 1.9×10 -4 | 2.1×10 5 | 1.27×10 6 |
| BHG | 1.17×10 -3 | 1.48×10 -3 | 0.79 | 2.88??? | 8.94×10 -6 | 3.22×10 5 | 4.08×10 5 |
Embodiment 4
The application of variant enzyme BHG in the polypeptide ligation
In this embodiment, variant enzyme BHG is used for synthetic (the fragment coupling synthesis of calcitonin technology of 24+8) of thyrocalcitonin, detailed process is as follows:
12.7mg fragment I (iNoc-CGNLSTCMLGTY) is dissolved in 500ul CH3CN, Dropwise 5 00ul acetic anhydride under 0 ℃ of stirring, continue to stir 1 hour, lyophilize, add the dissolving of 200ul DMF and 600ul phosphoric acid buffer (pH7.2), add 3.2mg fragment II (TQDFNKFHTFPQTAIGVGAP-amide) and 40ul variant enzyme BHG (50uM), stirring at room 3 hours.Mass spectrometry results shows that the molecular weight (3526) of product is consistent with theoretical value (3525.33).As seen, BHG has taking over a job property of polypeptide chain.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
(1) general information:
(i) applicant: Shanghai Research Center of Biotechnology
(ii) denomination of invention: stable polypeptide ligase enzyme and method for making and purposes in the non-aqueous media
(iii) sequence number: 3
(2) information of SEQ ID NO.1
(i) sequence signature
(A) length: 1146 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
( xi ) ：SEQ ID NO.1：GTGAGAAGCA AAAAATTGTG GATCAGCTTG TTGTTTGCGT TAACGTTAAT CTTTACGATG 60GCGTTCAGCA ACATGTCTGC GCAGGCTGCC GGAAAAAGCA GTACAGAAAA GAAATACATT 120GTTGGATTTA AACAGACAAT GAGTGCCATG AGTTCCGCCA AGAAAAAGGA TGTTATTTCT 180GAAAAAGGCG GAAAGGTTCA AAAGCAATTT AAGTATGTTA ACGCGGCCGC AGCAACATTG 240GATGAAAAAG CTGTAAAAGA ATTGAAAAAA GATCCGAGCG TTGCATATGT GGAAGAAGAT 300CATATTGCAC ATGAATATGC GCAATCTGTT CCTTATGGCA TTTCTCAAAT TAAAGCGCCG 360GCTCTTCACT CTCAAGGTTA CACAGGCTCT AACGTAAAAG TAGCTGTTAT CGACAGCGGA 420ATTGACTCTT CTCATCCTGA CTTAAACGTC AGAGGCGGAC GAAGCTTCGT ACCTTCTGAA 480ACAAACCCAT ACCAGGACGG CAGTTCTCAC GGTACGCATG TAGCCGGTAC GATTGCCGCT 540CTTAATAACT CAATCGGTGT TCTGGGCGTA CGGCCAAGCG CATCGTTATA TGCAGTAAAA 600GTGCTTGATT CAACAGGAAG CGGCCAATAT AGCTGGATTA TTAACGGCAT TGAGTGGGCC 660ATTTCCAACT CTATGGATGT TATTAACATG AGCCTTGGCG GACCTTCTGG TTCTACAGCG 720CTGAAAACAG TCGTTGATAA AGCCGTTTCC AGCGGTATCG TCGTTGCTGC CGCTGCCGGA 780AACGAAGGTT CGTCCGGAAG CTCAAGCACA GTCGGCTACC CTGCAAAATA TCCTTCTACT 840ATTGCAGTAG GTGCGGTAAA CAGCAGCAAC CAAAGAGCTT CATTCTCAAG CGCAGGTTCT 900GAGCTTGATG TGATGGCTCC TGGCGTATCC ATCCAAAGCA CACTTCCTGG AGGCACTTAC 960GGCGCTTATA ACGGAACGTG CGCGGCGACT GCTCACGTTG CCGGAGCAGC AGCGTTAATT 1020CTTTCTAAGC ACCCGACTTG GACAAACGCG CAAGTCCGTG ATCGTTTAGA AAGCACTGCA 1080ACATATCTTG GAAACTCTTT CTACTATGGA AAAGGGTTAA TCAACGTACA AGCAGCTGCA 1140CAATAA 1146
(2) information of SEQ ID NO.2:
(i) sequence signature:
(A) length: 275 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ii) molecule type: polypeptide
(xi) sequence description: SEQ ID NO.2:AQSVPYGISQ IKAPALHSQG YTGSNVKVAV IDSGIDSSHP DLNVRGGASF 50VPSETNPYQD GSSHGTHVAG TIAALNNSIG VLGVSPSASL YAVKVLDSTG 100SGQYSWIING IEWAISNSMD VINMSLGGPS GSTALKTVVD KAVSSGIVVA 150AAAGNEGSSG SSSTVGYPAK YPSTIAVGAV NSSNQRASFS SAGSELDVMA 200PGVSIQSTLP GGTYGAYNGT CAATAHVAGA AALILSKHPT WTNAQVRDRL 250ESTATYLGNS FYYGKGLINV QAAAQ 275
(2) information of SEQ ID NO.3:
(i) sequence signature:
(A) length: 275 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ii) molecule type: polypeptide
(xi) sequence description: SEQ ID NO.3:AQSVPYGISQ IKAPALHSQG YTGSNVKVAV IDSGIDSSHP DLNVRGGASF 50VPSETNPYQD GSSHGTHVAG TIAALNNSIG VLGVSPSASL YAVKVLDSTG 100SGQYSWIING IEWAISNNMD VINMSLGGPS GSTALKTVVD KAVSSGIVVA 150AAAGNEGSSG SSSTVGYPAK YPSTIAVGAV NSSNQRASFS SAGSELDVMA 200PGVSIQSTLP GGTYGAYNGT SMATPHVAGA AALILSKHPT WTNAQVRDRL 250ESTATYLGNS FYYGKGLINV QAAAQ 275
Claims (12)
1. polypeptide ligase enzyme, it is the variant enzyme of subtilisin E, it is characterized in that, it contains Ser221Cys, Met222Ala and Pro225Ala sudden change.
2. polypeptide ligase enzyme as claimed in claim 1 is characterized in that, it contains the sudden change that is selected from down group:
Asn118Ser, and Ser24His/Lys27Asp/Ser236Cys.
3. polypeptide ligase enzyme as claimed in claim 2 is characterized in that it comprises variant enzyme BHG and BW.
4. polypeptide ligase enzyme as claimed in claim 1 is characterized in that, it has the aminoacid sequence shown in the SEQ ID NO.2.
5. a separated DNA sequence is characterized in that, the described polypeptide ligase enzyme of its coding claim 1.
6. dna sequence dna as claimed in claim 5 is characterized in that, it comprises the sequence that is selected from down group: SEQ ID NO.1.
7. an expression vector is characterized in that, it contains the described dna sequence dna of claim 5.
8. a host cell is characterized in that, it is transformed by the described expression vector of claim 7.
9. host cell as claimed in claim 8 is characterized in that, it is Bacillus subtilus BHG (AprE) CCTCC No.M98022.
10. method of producing the described polypeptide ligase enzyme of claim 1 is characterized in that the method comprising the steps of:
(a) will the encode dna sequence dna of this polypeptide ligase enzyme operationally is connected in expression regulation sequence, forms polypeptide and connects the expression of enzymes carrier;
(b) expression vector in the step (a) is transformed into host cell;
(c) under the condition that is fit to this polypeptide ligase enzyme of expression, cultivate, thereby give expression to this polypeptide ligase enzyme;
(d) separation and purification goes out this polypeptide ligase enzyme.
11. method as claimed in claim 10, it is characterized in that, separation and purification condition used in step (c) is: the supernatant 30%-60% ammonium sulfate precipitation that will ferment, throw out dissolves after DEAE-Dextran A-25 post and Acrylex P-150 post with the phosphoric acid buffer of pH6.0-7.4.
12. the purposes of polypeptide ligase enzyme as claimed in claim 1 is characterized in that, it is used to the polypeptide ligation in the non-aqueous media.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 98126744 CN1258744A (en) | 1998-12-31 | 1998-12-31 | Polypeptide ligase being stable in non-water medium and its preparation and application |
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|---|---|---|---|
| CN 98126744 CN1258744A (en) | 1998-12-31 | 1998-12-31 | Polypeptide ligase being stable in non-water medium and its preparation and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101218343B (en) * | 2005-07-08 | 2013-11-06 | 诺维信公司 | subtilisin variant |
| CN105874067A (en) * | 2013-06-27 | 2016-08-17 | 诺维信公司 | Subtilase variants and polynucleotides encoding same |
| CN119391658A (en) * | 2024-10-30 | 2025-02-07 | 天津凯莱英生物科技有限公司 | Polypeptide ligase mutant and method for preparing polypeptide |
-
1998
- 1998-12-31 CN CN 98126744 patent/CN1258744A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101218343B (en) * | 2005-07-08 | 2013-11-06 | 诺维信公司 | subtilisin variant |
| CN105874067A (en) * | 2013-06-27 | 2016-08-17 | 诺维信公司 | Subtilase variants and polynucleotides encoding same |
| CN119391658A (en) * | 2024-10-30 | 2025-02-07 | 天津凯莱英生物科技有限公司 | Polypeptide ligase mutant and method for preparing polypeptide |
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