CN1834246A - Expression control of TrxA, encoding zone gene, its prepn. and uses - Google Patents
Expression control of TrxA, encoding zone gene, its prepn. and uses Download PDFInfo
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- CN1834246A CN1834246A CN 200510046034 CN200510046034A CN1834246A CN 1834246 A CN1834246 A CN 1834246A CN 200510046034 CN200510046034 CN 200510046034 CN 200510046034 A CN200510046034 A CN 200510046034A CN 1834246 A CN1834246 A CN 1834246A
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Abstract
This invention relates to the manufacture and application of the expression regulating and encoding region gene of Escherichia coli thioredoxin (TrxA). The gene is manufactured by: cloning the expression regulating and encoding region of TrxA by PCR technique that has the basic group sequence in SEQ ID No.1, and cloning it the the upstream of pET-28a promoter to promote the soluble expression of heterologous protein in Escherichia coli. The results show that TrxA and heterologous protein can combine to coexpress in non-fusion mode in Escherichia coli BL21 (DE3), and can promote the solubility of heterologous protein.
Description
Technical field
The present invention relates to gene recombination and protein expression field, is a kind of TrxA expression regulation and coding region gene and preparation and application specifically, has made up the prokaryotic expression carrier that a kind of TrxA of utilization promotes the foreign protein solubility expression.
Background technology
Escherichia coli thioredoxin has the function of catalytic proteins disulfide bond reduction as a kind of redox protein, can help protein correctly folding.Experimental results show that the upstream of TrxA gene fusion expression, can increase the solubility of foreign protein in foreign gene.But the foreign protein of expressing is because aminoterminal has merged bigger TrxA albumen, will cause certain influence to himself biological function, must use special polypeptidase (as enteropeptidase) to downcut TrxA. and cutting efficiency very low.Chinese scholars replaces the TrxA fusion rotein with the method for the free coexpression TrxA of different plasmids in recent years.Utilize TrxA to have the characteristics of similar molecular chaperones,, increased the solubility of exogenous protein expression product significantly expression plasmid and the same host bacterium of TrxA expression plasmid cotransformation and the coexpression of foreign protein.But because the uncompatibility of plasmid makes the use of this method be subjected to certain limitation.And the method for the free TrxA of coexpression still is not reported in same plasmid.
Summary of the invention
The objective of the invention is to make up a kind of TrxA expression regulation and coding region gene and preparation and application, obtain a kind of novel prokaryotic expression carrier that utilizes the free coexpression of TrxA to promote foreign protein solubility expression in intestinal bacteria.
For achieving the above object, the technical solution used in the present invention is:
Expression regulation and the coding region gene of clone TrxA, comprise SphI and BglII restriction endonuclease sites, T7 promotor (T7 promoter), lactose operon (Lac Operator), ribosome bind site (ribosome binding site, tbs) and mature T rxA protein-coding region and end the fusion gene of son, it has base sequence among the sequence table SEQ ID NO:1;
The regulation and control of TrxA and the preparation method of coding region gene are template with expression vector pET-32a gene, adopt primer
TrxA-F，5’-CGT?GCA?TGC?TAA?TAC?GAC?TCA?CTA?TAG-3’：
TrxA-R，5’-ATG?AGA?TCT?TTA?GGC?CAG?GTT?AGC?GTC-3’。
Amplify expression regulation and the coding region gene fragment of TrxA by round pcr;
The TrxA expression regulation and the coding region gene base sequence that clone are as follows: SEQ ID NO:1GCATGC
TAAT ACGACTCACT ATAGG 
40
Promoter Lac?Operator
CCTCTAGAAA TAATTTTGTT?TAACTTTAA
80
RBS CDS
TGACGACAGT TTTGACACGG ATGTACTCAA?AGCGGACGGG?160
GCGATCCTCG TCGATTTCTG GGCAGAGTGG?TGCGGTCCGT?200
GCAAAATGAT CGCCCCGATT CTGGATGAAA?TCGCTGACGA?240
ATATCAGGGC AAACTGACCG TTGCAAAACT?GAACATCGAT 280
CAAAACCCTG GCACTGCGCC GAAATATGGC?ATCCGTGGTA?320
TCCCGACTCT GCTGCTGTTC AAAAACGGTG?AAGTGGCGGC?360
AACCAAAGTG GGTGCACTGT CTAAAGGTCA?GTTGAAAGAG?400
TTCCTCGACG CTAACCTGGC CTAAAGATCT 430
Utilize the enzyme of restriction endonuclease SphI and BglII to cut interconnection technique, the TrxA expression regulation and the coding region gene (base sequence among the sequence table SEQ ID NO:1) of cloning is integrated into upstream, expression vector pET-28a clonal expression district (substituting the base sequences of 401 to 598 of former pET28-a expression vectors); Make in same plasmid vector, utilize TrxA and foreign protein with free but not merge the form coexpression and promote the foreign protein solubility expression.
The present invention has following advantage:
1. the present invention adopts the method for coexpression TrxA and foreign protein in the simple substance grain, has overcome the problem of plasmid incompatibility, has simplified the operation that plasmid transforms.
2. the mode of utilizing at present the short foreign protein solubility expression of TrxA both at home and abroad all is to merge coexpression or the free coexpression of different plasmid with plasmid; The present invention adopts and to make TrxA albumen and the strategy (with plasmid free coexpression) of foreign protein with the free coexpression of non-fusion form, makes the foreign protein of expression not be subjected to that TrxA is proteic to be influenced, and the polypeptidase after also need not to express cuts.
3. the present invention is artificial constructed a kind of novel prokaryotic expression carrier trxA-pET-28a, utilize TrxA to have the characteristics of similar molecular chaperones, this carrier can promote to fold the correct of the foreign protein of expression in escherichia coli, improves the solubility expression level, reduces insoluble inclusion body expression amount.
4. for the new expression vector of embodiment preparation, the present invention utilizes the expression of two identical promotors (T7promoter) regulation and control TrxA and foreign protein, can only use a kind of inductor to induce, and the expression that makes TrxA and foreign protein is carried out synchronously, and (TrxA expresses the upstream of district in the exogenous protein expression district, introduce new restriction enzyme site and end son), realize coexpression.The TrxA of coexpression can obviously promote the solubility expression of foreign protein single-chain antibody ML3.9 (scFv-ML), 3-hydroxy-benzoic acid-6-monooxygenase (3HBA), and obviously reduces the inclusion body expression of Enteromycin C 2 (SEC2), tubercule bacillus helicase A subunit (GYRA).The structure of this new expression vector will help gene engineering method and prepare various solubility foreign proteins.
Description of drawings
Fig. 1 is that the TrxA expression regulation of pcr amplification and coding region gene are with 1.2% agarose gel electrophoresis analytical results figure, wherein: the pcr amplification product of 1.trxA; 2.DL2000 dna molecular amount standard;
Fig. 2 is the 12%SDS-PAGE electrophoretic analysis figure of the short scFv-ML3.9 solubility expression of TrxA of free coexpression, wherein: 1. inductive pET-28a-ml positive colony not; 2. the full cell expressing of inductive pET-28a-ml positive colony; 3. inductive pET-28a-ml positive colony solubility expression; 4. inductive pET-28a-ml positive colony inclusion body is expressed; 5. inductive trxA-pET-28a-ml positive colony not; 6. the full cell expressing of inductive trxA-pET-28a-ml positive colony; 7. inductive trxA-pET-28a-ml positive colony solubility expression; 8. inductive trxA-pET-28a-ml positive colony inclusion body is expressed; 9. molecular weight of albumen standard;
Fig. 3 is the 12%SDS-PAGE electrophoretic analysis figure of the short 3HBA solubility expression of TrxA of free coexpression, wherein: 1. inductive pET-28a-3hba positive colony not; 2. the full cell expressing of inductive pET-28a-3hba positive colony; 3. inductive pET-28a-3hba positive colony solubility expression; 4. inductive pET-28a-3hba positive colony inclusion body is expressed; 5. inductive trxA-pET-28a-3hba positive colony not; 6. the full cell expressing of inductive trxA-pET-28a-3hba positive colony; 7. inductive trxA-pET-28a-3hba positive colony solubility expression; 8. inductive trxA-pET-28a-3hba positive colony inclusion body is expressed; 9. molecular weight of albumen standard;
Fig. 4 is the 12%SDS-PAGE electrophoretic analysis figure of the short SEC2 solubility expression of TrxA of free coexpression, wherein: 1. inductive pET-28a-sec2 positive colony not; 2. the full cell expressing of inductive pET-28a-sec2 positive colony; 3. inductive pET-28a-sec2 positive colony solubility expression; 4. inductive pET-28a-sec2 positive colony inclusion body is expressed; 5. inductive trxA-pET-28a-sec2 positive colony not; 6. the full cell expressing of inductive trxA-pET-28a-sec2 positive colony; 7. inductive trxA-pET-28a-sec2 positive colony solubility expression; 8. inductive trxA-pET-28a-sec2 positive colony inclusion body is expressed; 9. molecular weight of albumen standard;
Fig. 5 is the 12%SDS-PAGE electrophoretic analysis figure of the short GYRA solubility expression of TrxA of free coexpression, wherein: 1. inductive pET-28a-gyra positive colony not; 2. the full cell expressing of inductive pET-28a-gyra positive colony; 3. inductive pET-28a-gyra positive colony solubility expression; 4. inductive pET-28a-gyra positive colony inclusion body is expressed; 5. inductive trxA-pET-28a-gyra positive colony not; 6. the full cell expressing of inductive trxA-pET-28a-gyra positive colony; 7. inductive trxA-pET-28a-gyra positive colony solubility expression; 8. inductive trxA-pET-28a-gyra positive colony inclusion body is expressed; 9. molecular weight of albumen standard;
Embodiment
The expression regulation of TrxA and the preparation of coding region gene, it has the base sequence shown in the sequence table SEQ ID NO:1:
(1) information of SEQ ID NO.1 (referring to sequence table)
(a) sequence signature:
* length: 430 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: carrier pET32-a plasmid DNA
(2) pcr amplification of the regulation and control of TrxA and coding region gene
1) PCR design of primers and reaction conditions:
According to the dna sequence dna of TrxA gene order and pET32-a, design one group of primer:
Forward primer TrxA-F:5 '-CGT GCA TGC TAA TAC GAC TCA CTA TAG-3 ';
Reverse primer TrxA-R:5 '-ATG AGA TCT TTA GGC CAG GTT AGC GTC-3 '
The PCR reaction system is: 10 * Pyrobest buffer, 5 μ l, dNTP 250 μ mol, 0.02%BSA 2 μ l, each 25pmol of upstream and downstream primer, template pET32-a plasmid DNA 0.1 μ g, PyrobestDNA polysaccharase 2U, aseptic ultrapure water polishing volume to 50 μ l.
The PCR reaction conditions is:
95 ℃ of fs, 5 minutes;
94 ℃ of subordinate phase, 45 seconds; 55 ℃, 45 seconds; 72 ℃, 35 seconds; Totally 30 circulations;
72 ℃ of phase IIIs, 10 minutes;
2) recovery of PCR product: pcr amplification product is through 1.2% agarose gel electrophoresis analysis and separation (accompanying drawing 1), and downcutting size is the purpose band of 430bp, reclaims the test kit working instructions by the clean biochemical technology of Hangzhou Wei Te company limited glue and carries out the glue recovery;
3) double digestion of recovery after product: the PCR product after the recovery fully digests through restriction endonuclease SphI and BglII, and digestion product is through 1.2% agarose gel electrophoresis analysis, and glue reclaims once more.
The structure of new expression vector trxA-pET-28a
(1) double digestion of pET-28a carrier: the U.S.'s pET-28a of Novagen company vector plasmid DNA is fully digested through restriction endonuclease SphI and BglII, and digestion product is through 0.8% agarose gel electrophoresis analysis, and glue reclaims the big fragment of purifying 5100bp
(2) enzyme is cut the connection and the conversion of product: will mix through the PCR of double digestion product fragment and the big fragment of the pET-28a carrier molar ratio with 3: 1, and spend the night with 16 ℃ of connections of T4DNA ligase enzyme, and be built into expression vector trxA-pET-28a.(conversion operation is pressed F. Ao Sibai, R. Brunt, R.E. James Kingston to connect product Transformed E .coli BL21 (DE3) competent cell, D.D. Moore, J.G. Sai Deman, J.A. Smith, K. Si Telaer " fine works molecular biology experiment guide " USA New York JohnWiley﹠amp; The nineteen ninety-five third edition P39-40 of Sons press), with kalamycin resistance screening transformant.
(3) checking of expression vector trxA-pET-28a: the positive transformant of selecting is spread cultivation, (the plasmid DNA extracting method is pressed J. Sa nurse Brooker to extract plasmid DNA, E.F. Flitch, T. Manny A Disi " molecular cloning experiment guide " cold spring port, the USA New York calendar year 2001 third edition P27-30 of press), identify through SphI and BglII double digestion, and to identify that correct recombinant clone plasmid is a template, be primer with T7 promotor and terminator, confirm with the terminal cessation method order-checking of the two deoxidations of Sanger.
The practical application of new expression vector trxA-pET-28a and effect assessment
(1) exogenous genetic fragment is connected into new expression vector trxA-pET-28a: trxA-pET-28a plasmid DNA and the pET-28a-ml plasmid DNA that contains single-chain antibody ML3.9 gene are used Nco I, Not I double digestion respectively, through 1.0% agarose gel electrophoresis, glue reclaims scFv-ml fragment and the trxA-pET-28a plasmid 5500bp fragment that test kit reclaims 770bp, spend the night with 16 ℃ of connections of T4DNA ligase enzyme, make up single-chain antibody ML3.9 and TrxA co-expression carrier trxA-pET-28a-ml.Connect product Transformed E .coli BL21 (DE3) competent cell.With kalamycin resistance screening transformant, select recombinant clone and spread cultivation, extract plasmid DNA, identify correct recombinant clone through Nco I, Not I double digestion.Make up 3-hydroxy-benzoic acid-6-monooxygenase (3HBA) and TrxA co-expression carrier trxA-pET-28a-3hba with quadrat method with Nhe I, EcoR I double digestion; Make up Enteromycin C 2 (SEC2) and TrxA co-expression carrier trxA-pET-28a-sec2 with EcoRI, XhoI double digestion; Make up tubercule bacillus helicase A subunit (GYRA) and TrxA co-expression carrier trxA-pET-28a-gyra with EcoRI, XhoI double digestion
(2) coexpression of TrxA and foreign protein: respectively inoculation transformed each recombinant plasmid the single bacterium colony of BL21 (DE3) in the liquid LB substratum that contains 40 μ g/ml kantlex, the activation culture of spending the night, with 1% inoculum size switching next stage, 37 ℃ of shaking tables are cultured to OD
600Be 0.6, add the IPTG of final concentration 1.0mmol/L, 30 ℃ of abduction deliverings 6 hours
(3) analysis of the short foreign protein solubility expression of TrxA: centrifugal collection thalline, PBS buffer solution for cleaning thalline with the 1/10 former culture volume of pH7.4 is once also resuspended, become limpid in 0 ℃ of ultrasonication until bacterium liquid, centrifugal 20 minutes of 12000r/min, get supernatant, with the resuspended precipitation of PBS damping fluid of 1/30 former culture volume.Get respectively after ultrasonic gross sample and centrifugal after the resuspended liquid of last cleer and peaceful precipitation that obtains, (buffer formulation and treatment process are pressed Wang Jiazheng with the processing of 5 * sample-loading buffer, model bright " protein technical manual " 2002 first version P81 and the P87 of Science Press), the 12%SDS-PAGE electrophoresis, coomassie brilliant blue staining is analyzed expression product, and thin layer chromatography scanner scans at wavelength 560nm place
The result: TrxA albumen, scFvML3.9 albumen, 3-HBA albumen, SEC2 albumen and the proteic molecular weight of GYRA are respectively 12kD, 36kD, 45kD, 31kD and 96kD, through inducing of the IPTG of 1.0mmol/L, clone's trxA gene obtains great expression, and the coexpression that can dissociate with the foreign gene in same plasmid vector.
With the effect of TrxA albumen coexpression under, the proteic solubility expression amount of scFv-ML3.9 albumen and 3-HBA all has remarkable increase (accompanying drawing 2,3), the proteic inclusion body of SEC2 albumen and GYRA is expressed and is significantly reduced (accompanying drawing 4,5), as seen, the free coexpression of TrxA has promoted the correct folding of foreign protein, reduce the formation of inclusion body, impelled albumen to exist with soluble form more.
SEQUENCE?LISTING
＜110〉Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences
＜120〉a kind of TrxA expression regulation and coding region gene and preparation and application altogether
<130>
<160>2
<170>PatentIn?version?3.1
<210>1
<211>430
<212>DNA
＜213〉carrier pET32-a plasmid DNA
<220>
<221>CDS
<222>(95)..(430)
<223>
<400>1
gcatgctaat?acgactcact?ataggggaat?tgtgagcgga?taacaatt?cc?cctctagaaa 60
taattttgtt?taactttaag?aaggagatat?acat?atg?agc?gat?aaa?att?att?cac 115
Met?Ser?Asp?Lys?Ile?Ile?His
1 5
ctg?act?gac?gac?agt?ttt?gac?acg?gat?gta?ctc?aaa?gcg?gac?ggg?gcg 163
Leu?Thr?Asp?Asp?Ser?Phe?Asp?Thr?Asp?Val?Leu?Lys?Ala?Asp?Gly?Ala
10 15 20
atc?ctc?gtc?gat?ttc?tgg?gca?gag?tgg?tgc?ggt?ccg?tgc?aaa?atg?atc 211
Ile?Leu?Val?Asp?Phe?Trp?Ala?Glu?Trp?Cys?Gly?Pro?Cys?Lys?Met?Ile
25 30 35
gcc?ccg?att?ctg?gat?gaa?atc?gct?gac?gaa?tat?cag?ggc?aaa?ctg?acc 259
Ala?Pro?Ile?Leu?Asp?Glu?Ile?Ala?Asp?Glu?Tyr?Gln?Gly?Lys?Leu?Thr
40 45 50 55
gtt?gca?aaa?ctg?aac?atc?gat?caa?aac?cct?ggc?act?gcg?ccg?aaa?tat 307
Val?Ala?Lys?Leu?Asn?Ile?Asp?Gln?Asn?Pro?Gly?Thr?Ala?Pro?Lys?Tyr
60 65 70
ggc?atc?cgt?ggt?atc?ccg?act?ctg?ctg?ctg?ttc?aaa?aac?ggt?gaa?gtg 355
Gly?Ile?Arg?Gly?Ile?Pro?Thr?Leu?Leu?Leu?Phe?Lys?Asn?Gly?Glu?Val
75 80 85
gcg?gca?acc?aaa?gtg?ggt?gca?ctg?tct?aaa?ggt?cag?ttg?aaa?gag?ttc 403
Ala?Ala?Thr?Lys?Val?Gly?Ala?Leu?Ser?Lys?Gly?Gln?Leu?Lys?Glu?Phe
90 95 100
ctc?gac?gct?aac?ctg?gcc?taa?aga?tct 430
Leu?Asp?Ala?Asn?Leu?Ala Arg?Ser
105 110
<210>2
<211>109
<212>PRT
＜213〉carrier pET32-a plasmid DNA
<400>2
Met?Ser?Asp?Lys?Ile?Ile?His?Leu?Thr?Asp?Asp?Ser?Phe?Asp?Thr?Asp
1 5 10 15
Val?Leu?Lys?Ala?Asp?Gly?Ala?Ile?Leu?Val?Asp?Phe?Trp?Ala?Glu?Trp
20 25 30
Cys?Gly?Pro?Cys?Lys?Met?Ile?Ala?Pro?Ile?Leu?Asp?Glu?Ile?Ala?Asp
35 40 45
Glu?Tyr?Gln?Gly?Lys?Leu?Thr?Val?Ala?Lys?Leu?Asn?Ile?Asp?Gln?Asn
50 55 60
Pro?Gly?Thr?Ala?Pro?Lys?Tyr?Gly?Ile?Arg?Gly?Ile?Pro?Thr?Leu?Leu
65 70 75 80
Leu?Phe?Lys?Asn?Gly?Glu?Val?Ala?Ala?Thr?Lys?Val?Gly?Ala?Leu?Ser
85 90 95
Lys?Gly?Gln?Leu?Lys?Glu?Phe?Leu?Asp?Ala?Asn?Leu?Ala
100 105
Claims (3)
1. TrxA expression regulation and coding region gene is characterized in that: have base sequence among the sequence table SEQ IDNO:1.
2. the preparation of TrxA expression regulation and coding region gene, it is characterized in that: with expression vector pET-32a gene is template, adopts primer
TrxA-F，5’-CGT?GCA?TGC?TAA?TAC?GAC?TCA?CTA?TAG-3’；
TrxA-R，5’-ATG?AGA?TCT?TTA?GGC?CAG?GTT?AGC?GTC-3’
Amplify gene fragment with base sequence among the sequence table SEQ ID NO:1 with round pcr.
3. the application of TrxA expression regulation and coding region gene is characterized in that: base sequence among the sequence table SEQ ID NO:1 is cloned into 401 to 598 of pET28-a expression vectors, is built into the prokaryotic expression carrier TrxA-pET28-a of short solubility expression.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510046034 CN1834246A (en) | 2005-03-16 | 2005-03-16 | Expression control of TrxA, encoding zone gene, its prepn. and uses |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510046034 CN1834246A (en) | 2005-03-16 | 2005-03-16 | Expression control of TrxA, encoding zone gene, its prepn. and uses |
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| Publication Number | Publication Date |
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| CN1834246A true CN1834246A (en) | 2006-09-20 |
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| CN 200510046034 Pending CN1834246A (en) | 2005-03-16 | 2005-03-16 | Expression control of TrxA, encoding zone gene, its prepn. and uses |
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| Country | Link |
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| CN (1) | CN1834246A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928285A (en) * | 2014-03-21 | 2015-09-23 | 中国科学院沈阳应用生态研究所 | Vector cloning and expression region gene and protein secretion type mammal cell expression vector |
| CN108300728A (en) * | 2017-01-12 | 2018-07-20 | 中国科学院沈阳应用生态研究所 | A kind of bis- dissolutions expression sequence labels of TrxA and SUMO and application |
-
2005
- 2005-03-16 CN CN 200510046034 patent/CN1834246A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928285A (en) * | 2014-03-21 | 2015-09-23 | 中国科学院沈阳应用生态研究所 | Vector cloning and expression region gene and protein secretion type mammal cell expression vector |
| CN108300728A (en) * | 2017-01-12 | 2018-07-20 | 中国科学院沈阳应用生态研究所 | A kind of bis- dissolutions expression sequence labels of TrxA and SUMO and application |
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