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Escherichia coli soluble expression vector capable of efficiently obtaining recombinant protein

A technology for expressing vectors and recombinant proteins, applied in the direction of vectors, nucleic acid vectors, viruses/phages, etc., can solve the problems of low expression of foreign proteins, many operation steps, and low purity, and achieve high application value, simplified operation steps, The effect of improving purity

Pending Publication Date: 2017-10-20
SHENYANG PHARMA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The invention provides an Escherichia coli soluble expression vector capable of efficiently obtaining recombinant proteins and a construction method thereof, and the expression of exogenous proteins by using the vector overcomes the low expression level and low purity of exogenous proteins existing in the prior art , many operating steps, defects expressed in the form of inclusion bodies

Method used

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  • Escherichia coli soluble expression vector capable of efficiently obtaining recombinant protein
  • Escherichia coli soluble expression vector capable of efficiently obtaining recombinant protein
  • Escherichia coli soluble expression vector capable of efficiently obtaining recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: soluble expression vector pSYPU-IH constructs

[0032] Strain: E.coli BL21(DE3) is preserved in our laboratory

[0033] Primers:

[0034] LG4S-F1: 5'-GGTGGCGGCGGTAGTGGCGGCGGTGGTAGTAAAATCGAAGAAGGTAAACTGACAAATCC-3'

[0035] LG4S-F2: 5'-CGCCATATG(CAT) 5 CACGGTGGCGGCGGTAGTGGCGGCGGTGGTAGT-3'

[0036] LG4S-R1: 5'-CGCGGATCCGAATTCGAGCTCGCTCTTCCGTTGTGTACAATGAT-3'

[0037] ProH-F1: 5'-CGCGAATTCTAAGAAGGAGATATACATATGAGCGATAAAATTATTCACCTGAC-3'

[0038] ProH-R1: 5'-ACTACCACCGCCGCCACTACCGCCGCCACCGCTGCTGGCCAGGTTAGC-3'

[0039] ProH-F2: 5'-GGTGGCGGCGGTAGTGGCGGCGGTGGTAGTATGAGCGATAAAATTATTCACCTGAC-3'

[0040] ProH-R2: 5'-CGCGGATCCTTAGCTATGATGATGATGATGGTGGCTGCTGGCCAGGTTAG-3'

[0041] ProH-F: 5'-CGCGAATTCTAAGAAGGAGATATACATATGAGCGATAAAATTATTCACCTGAC-3'

[0042] ProH-R: 5'-CGCGGATCCTTAGCTATGATGATGATGATGGTGGCTGCTGGCCAGGTTAG-3'

[0043] ANGP-F1: 5'-GGTGGTTGCTCTTCCAACGATGGATATATAAGAGGAAGTAACG-3'

[0044] ANGP-R1: 5'-CGCGAATTCTTACTTTTTGCCACCGCATGTATTACT-3'

[0045] 1.1 (h...

Embodiment 2

[0062] Example 2: Soluble expression, separation and purification of recombinant scorpion venom active peptide ANGP

[0063] 2.1 Construction of recombinant plasmid pSYPU-IH-ANGP

[0064] Using pET28a-ANGP as a template, with Bsp I and EcoR I restriction endonuclease recognition site ANGP F / R as primers, PCR amplification of ANGP gene (see appendix) Figure 2-1 ). The ANGP gene was digested with EcoR I at 37°C for 2 hr, separated by 1.5% agarose gel electrophoresis, and the ANGP gene was recovered by gel (see appendix). Figure 2-2 ), the recovered product of ANGP gene after being digested by EcoR I single enzyme was subjected to Bsp I single enzyme digestion, and the Bsp I single enzyme digestion of ANGP gene was performed at 50°C for 1 hr. The recombinants were screened by LB-Amp and verified by sequencing.

[0065] 2.2 Induction and expression of recombinant plasmid pSYPU-IH-ANGP

[0066] Take the recombinant plasmid pSYPU-IH-ANGP, heat-transform E. coli BL21 (DE3) comp...

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Abstract

The invention discloses an escherichia coli soluble expression vector capable of efficiently obtaining recombinant protein. A hexahistidine label-flexible linker peptide-chitin binding domain-peptide-containing complex coding gene and a thioredoxin-hexahistidine label complex coding gene are placed in the same vector, the characteristics that the peptide-containing protein can realize self-cutting under the condition of change of temperature and pH and the thioredoxin can promote correct folding of protein containing more disulfide bonds are used, through affinity mediation of a hexahistidine label or a chitin binding domain, by using an affinity chromatographymethod, the target protein purificationefficiency and purity can be greatly improved, the problem that Escherichia coli gene engineering expression products form inclusion bodies easily is solved at a low cost, the application value is high, and the application range is wide.

Description

technical field [0001] The invention relates to a prokaryotic expression system, in particular to a soluble expression vector capable of obtaining recombinant protein with high efficiency and low cost. Background technique [0002] In the research of genetically engineered drugs, Escherichia coli has the advantages of clear genetic background, fast reproduction, low cost, high expression level, and easy operation, but there are inevitably certain shortcomings. Although people have developed a variety of expression systems to overcome the shortcomings of Escherichia coli expression products such as endotoxin, lack of post-translational modifications, and easy folding errors during high expression, the Escherichia coli expression system is still an important tool for gene expression. tool. [0003] When Escherichia coli is used to express foreign genes at a high level, especially when the expression products are rich in disulfide bonds, inclusion bodies are easy to form, and ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/65C12N15/66C12N15/67
CPCC12N15/65C12N15/66C12N15/67C12N15/70C12N2800/101C12N2840/002C12N2840/44
Inventor 宋永波张景海
Owner SHENYANG PHARMA UNIVERSITY
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