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TrxA and SUMO double-solubilized expression tag sequence and application thereof

A technology for expressing tag sequences and solubilizing tags, applied in recombinant DNA technology, biochemical equipment and methods, using vectors to introduce foreign genetic material, etc. The effect is not very satisfactory and other problems, to save time and cost of research, promote correct folding, and accurate enzyme digestion

Inactive Publication Date: 2018-07-20
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

[0002] Since the prokaryotic expression system lacks an appropriate post-translational processing mechanism, in the process of expressing foreign proteins in Escherichia coli as the host bacteria, insoluble inclusion bodies will be formed due to incorrect protein folding, which requires complex denaturation and renaturation treatment again. Difficulty expressing large amounts of soluble foreign protein
[0003] Escherichia coli thioredoxin TrxA has the function of catalyzing the reduction of protein disulfide bonds, which can help proteins to fold correctly. Co-expression can increase the solubility of foreign proteins, and there is an enterokinase site downstream of TrxA, which can be used after expression. Enzymatically cut Trx to ensure the expression of biological functions of foreign proteins, but its cutting efficiency is low, and the effect of solubilization is not ideal

Method used

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  • TrxA and SUMO double-solubilized expression tag sequence and application thereof
  • TrxA and SUMO double-solubilized expression tag sequence and application thereof
  • TrxA and SUMO double-solubilized expression tag sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The base sequence of the TrxA and SUMO dual pro-solution expression tag is as follows: SEQ ID NO: 1

[0028]

[0029] (1) Information of SEQ ID NO: 1 (see sequence listing)

[0030] (a) Sequence features:

[0031] *Length: 750bp

[0032] *Type: nucleic acid

[0033] * Chain type: double chain

[0034] *Topology: Linear

[0035] (b) Molecular type: cDNA

[0036] (c) Assumption: No

[0037] (d) Antisense: No

[0038] (e) Original source: Synthetic

[0039] (2) PCR amplification of SUMO gene

[0040] (a) PCR primer design and reaction conditions:

[0041] A set of primers were designed according to the SUMO gene sequence and the DNA sequence of pET32a:

[0042] Forward primer: 5'-CGGGGTACCATGTCGGACTCAGAAGTCAAT-3' (synthesized by Beijing Huada Gene Company)

[0043] Reverse primer: 5'-CAGTCCATGGCACCAATCTGTTCTCTGTGAGC-3' (synthesized by Beijing Huada Gene Company)

[0044] The PCR reaction system was: 10xPyrobest buffer (purchased from Dalian Bao Biological Co...

Embodiment 2

[0052] Fusion connection of SUMO sequence and TrxA sequence

[0053] (1) Double enzyme digestion of the pET32a vector: The pET32a vector plasmid DNA (purchased from Novagen) was double-digested with restriction endonucleases Kpn I and Nco I, and the digested product was analyzed by 1.5% agarose gel electrophoresis, and the gel was recovered Large fragments of DNA.

[0054](2) Ligation and transformation of digested products: Mix the double-digested SUMO PCR product fragment and pET32a vector at a molar ratio of 3:1, and use T4 DNA ligase (purchased from Dalian Bao Biological Company) to ligate overnight at 16°C , to construct a new expression vector pET-32a-SUMO. The ligation product was transformed into Escherichia coli competent cell DH5α (purchased from Dalian Bao Biology Co., Ltd.) (transformation operation was carried out according to Page 39-40 of the third edition of "Molecular Biology Experiment Guide" edited by F. Osper et al.), using ampicillin Transformants were s...

Embodiment 3

[0057] Application and effect evaluation of TrxA and SUMO dual pro-solution expression tag sequence

[0058] (1) Ligate the exogenous gene scFv-Sag into the gene containing TrxA and SUMO

[0059] The expression vector pET-32a-SUMO of the dual solubilizing expression tag sequence: the pET-32a-SUMO plasmid DNA and the plasmid pET-32a-scFv-Sag containing scFv-SAg (see the reference for the construction process: Xu Mingkai et al. 2006. Fusionimmunotoxin anti-HER-2-scFv-SEC2 expressed in E.coli with an improved expression vector pASK75-EX: its construction and function. Advances in Biochemistry and Biophysics (English Edition). 33(8):781-788.) DNA respectively Using Nco I and Xho I (purchased from Dalian Bao Biological Co., Ltd.) double digestion, after 1.0% agarose gel electrophoresis, gel recovery of 1881bp scFv-SAg fragment and 6100bp pET-32a-SUMO plasmid fragment, with T4DNA ligase After ligation at 16°C overnight, the fusion protein expression vector pET-32a-SUMO-scFv-SAg was...

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Abstract

The invention relates to the fields of gene recombination and prokaryotic expression of proteins, and concretely relates to a double-solubilized expression tag sequence formed through fusing Escherichia coli thioredoxin TrxA and a small ubiquitin-like modifier SUMO, and an application thereof in a prokaryotic expression vector. An SUMO and TrxA double-solubilized expression tag has a base sequencerepresented by SEQ ID NO:1 in a sequence table. The sequence is applied to the upstream of the foreign protein encoding gene of the prokaryotic expression vector, and participates in fusion expression as a solubilizing tag together with a foreign gene in order to significantly promote the solubility of the expressed foreign protein and improve the recovery efficiency of a soluble foreign protein.

Description

technical field [0001] The present invention relates to the field of gene recombination and protein prokaryotic expression, specifically a dual prokaryotic expression tag, which is composed of Escherichia coli thioredoxin TrxA and small ubiquitin-like modification protein SUMO in series, which is used for prokaryotic expression vectors Upstream of foreign protein coding genes to promote correct folding of the expressed foreign protein and enhance solubility. Background technique [0002] Since the prokaryotic expression system lacks an appropriate post-translational processing mechanism, in the process of expressing foreign proteins in Escherichia coli as the host bacteria, insoluble inclusion bodies will be formed due to incorrect protein folding, which requires complex denaturation and renaturation treatment again. It is difficult to express large amounts of soluble foreign protein. [0003] Escherichia coli thioredoxin TrxA has the function of catalyzing the reduction of...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/65
CPCC12N15/65C12N15/70C12N2800/101C12N2800/70
Inventor 徐明恺宋宇博李永强李旭张惠文张成刚
Owner SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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