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Expression control of TrxA, encoding zone gene, its prepn. and uses

A technology of expression regulation and coding region, applied in DNA preparation, application, genetic engineering, etc., can solve the problems of plasmid incompatibility, usage restriction, etc., and achieve the effect of reducing expression amount, promoting correct folding, and improving soluble expression level.

Inactive Publication Date: 2006-09-20
SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the incompatibility of the plasmid, the use of this method is limited
However, the method of co-expressing free TrxA in the same plasmid has not been reported yet.

Method used

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  • Expression control of TrxA, encoding zone gene, its prepn. and uses
  • Expression control of TrxA, encoding zone gene, its prepn. and uses
  • Expression control of TrxA, encoding zone gene, its prepn. and uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The expression regulation of TrxA and the preparation of the coding region gene, which has the base sequence shown in the sequence table SEQ ID NO: 1:

[0036] (1) Information of SEQ ID NO.1 (see sequence listing)

[0037] (a) Sequence features:

[0038] * Length: 430 bp

[0039] *Type: nucleic acid

[0040] * Chain type: double chain

[0041] *Topology: Linear

[0042] (b) Molecular type: cDNA

[0043] (c) Assumption: No

[0044] (d) Antisense: No

[0045] (e) Original source: vector pET32-a plasmid DNA

[0046] (2) Regulation of TrxA and PCR amplification of genes in the coding region

[0047] 1) PCR primer design and reaction conditions:

[0048] According to the TrxA gene sequence and the DNA sequence of pET32-a, a set of primers were designed:

[0049] Forward primer TrxA-F: 5'-CGT GCA TGC TAA TAC GAC TCA CTA TAG-3';

[0050] Reverse primer TrxA-R: 5'-ATG AGA TCT TTA GGC CAG GTT AGC GTC-3'

[0051] The PCR reaction system was: 10×Pyrobest buffer 5 μl, dNT...

Embodiment 2

[0059] Construction of a New Expression Vector trxA-pET-28a

[0060] (1) Double enzyme digestion of the pET-28a vector: The DNA of the pET-28a vector plasmid from Novagen, USA was fully digested with restriction endonucleases SphI and BglII, the digested product was analyzed by 0.8% agarose gel electrophoresis, and the gel was recovered and purified 5100bp large fragment

[0061] (2) Ligation and transformation of enzyme-digested products: Mix the double-enzyme-digested PCR product fragment and the pET-28a vector large fragment at a molar ratio of 3:1, and ligate overnight at 16°C with T4 DNA ligase to construct an expression vector trxA-pET-28a. The ligation product was transformed into E.coli BL21 (DE3) competent cells (transformation operation according to F. Osper, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Ster Lal "Refined Molecular Biology Experiment Guide" New York John Wiley & Sons Publishing House, 1995 third edition P39-40), to screen transf...

Embodiment 3

[0064] Practical application and effect evaluation of new expression vector trxA-pET-28a

[0065] (1) Ligate the exogenous gene fragment into the novel expression vector trxA-pET-28a: the trxA-pET-28a plasmid DNA and the pET-28a-ml plasmid DNA containing the single-chain antibody ML3. I double enzyme digestion, 1.0% agarose gel electrophoresis, gel recovery kit recovery 770bp scFv-ml fragment and trxA-pET-28a plasmid 5500bp fragment, ligated overnight at 16°C with T4 DNA ligase to construct single-chain antibody ML3. 9 co-expression vector trxA-pET-28a-ml with TrxA. The ligation product was transformed into E.coli BL21(DE3) competent cells. The transformants were screened by kanamycin resistance, the recombinant clones were selected and expanded, the plasmid DNA was extracted, and the correct recombinant clones were identified by Nco I and Not I double enzyme digestion. In the same way, construct 3-hydroxybenzoic acid-6-monooxygenase (3HBA) and TrxA co-expression vector trxA...

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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 [0001] The invention relates to the fields of gene recombination and protein expression, specifically a TrxA expression regulation and coding region gene and its preparation and application, and constructs a prokaryotic expression vector that utilizes TrxA to promote the soluble expression of exogenous proteins. Background technique [0002] Escherichia coli thioredoxin, as a redox protein, has the function of catalyzing the reduction of protein disulfide bonds, which can help proteins to fold correctly. Experiments have proved that the fusion expression of TrxA gene in the upstream of exogenous gene can increase the solubility of exogenous protein. However, due to the large TrxA protein fused to the amino terminal of the expressed foreign protein, it will have a certain impact on its own biological function, and a special polypeptide enzyme (such as enterokinase) must be used to cut off TrxA. And the cutting efficiency is very low. In recent years, scholars...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/10C12N15/63C12N15/70C12N15/74
Inventor 徐明恺张成刚
Owner SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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