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SUMO and SUMO protease encoding gene and application thereof

A protease and gene technology, applied in the fields of genetic engineering and protein engineering, to achieve high expression, increase expression, and broaden application

Inactive Publication Date: 2017-02-22
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Currently, there is a lack of a highly expressed in Escherichia coli Application of SUMO and SUMO protease gene and its encoded protein

Method used

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  • SUMO and SUMO protease encoding gene and application thereof
  • SUMO and SUMO protease encoding gene and application thereof
  • SUMO and SUMO protease encoding gene and application thereof

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preparation example Construction

[0045] A kind of preparation method of the recombinant vector of pET28b-sumo (S) and pMF-ulp (S) of the present invention, comprises the steps:

[0046] (1) Artificially synthesizing the genes shown in SEQ ID NO: 1 and SEQ ID NO: 3 in the sequence table respectively;

[0047] (2) Construct the recombinant vectors of pET28b-sumo (S) and pMF-ulp (S) respectively; the pMF-ulp (S) recombinant vector is selective to the vector, and the present invention proves that ulp (S) is constructed in pET28b Most of the expression in the vector is inclusion body precipitation, while soluble expression can be achieved in the pMF vector.

[0048] The expression vector of the Saccharomyces cerevisiae gene sumo (S) is pET28b, the expression vector of the SUMO protease gene ulp (S) is a pMF vector, and the host bacterium is Escherichia coli DH5α;

[0049] Using DNA recombination technology, insert the sumo(S) gene shown in the sequence table SEQ ID NO: 1 into the Nde Ⅰ and BamH Ⅰ enzyme digestion...

Embodiment 1

[0064] Construction, expression, detection and expression measurement of sumo(S) and ulp(S) recombinant vectors

[0065] The sumo(S) gene and pET28b vector were digested with Nde Ⅰ and BamH Ⅰ, and the ulp(S) gene and pMF vector were digested with BamHI and HindⅢ. After gel recovery, the corresponding gene and vector were ligated with T4 DNA ligase at 16°C for 5 hours. , the ligation product was transferred into E. coli DH5α competent cells, after incubation and refolding, smear a resistant plate and culture at 37°C for 12 hours, then pick a single colony, and culture in 5mL Luria-Bertani medium at 37°C, 220rpm for 12 hours Plasmids were extracted and sequenced after enzyme digestion and verification.

[0066] The correctly sequenced pET28b-sumo(S) and pMF-ulp(S) plasmids were transferred into BL21(DE3) competent cells, the control plasmids were pET28b-sumo(wt) and pET28b-ulp(wt), and the control plasmids Transform into BL21(DE3) and Rosetta(DE3) competent cells, pick a single...

Embodiment 2

[0073] Construction and expression of ulp(S) recombinant vector containing six kinds of fusion tags

[0074] The six tags of dnaK, mbp, gst, tig, groEL, trx and pET28b-tevS were digested with Nde Ⅰ and Bgl Ⅱ and then ligated and transformed into DH5α competent cells. The vector and ulp(S) were digested with BamH Ⅰ and Hind Ⅲ and then ligated. After the transformation and digestion were verified, the six plasmids were transferred into BL21(DE3) competent cells. -Bertani medium 37 ℃, 220rpm shaking culture, bacteria grow to OD 600 At 0.6, add IPTG with a final concentration of 0.5mmol / L and induce at 28°C for 12h, then collect the bacteria and sonicate them, freeze and centrifuge the supernatant protein samples for SDS-PAGE electrophoresis. Such as image 3 shown. The results showed that the expression of ULP(S) with tag fusion was good.

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Abstract

The invention discloses a SUMO (small ubiquitin-related modifier) and SUMO protease encoding gene, encoding protein and application thereof. The invention relates to a Saccharomyces cerevisiae gene sumo (S), which is defined by the nucleotide sequence of SEQ ID No.1 in the sequence table. The encoding protein of the Saccharomyces cerevisiae gene sumo (S) is defined by the amino acid sequence of SEQ ID No.2 in the sequence table. The SUMO protease gene ulp (S) provided by the invention is defined by the nucleotide sequence of SEQ ID No.3 in the sequence table. The encoding protein of SUMO protease gene ulp (S) provided by the invention is defined by the amino acid sequence of SEQ ID No.4 in the sequence table. The invention can realize high expression of Saccharomyces cerevisiae sumo gene and SUMO protease ulp gene in Escherichia coli, specifically realize high expression in the commonly used expression strain BL21(DE3) of Escherichia coli, and ensures that the function is not affected.

Description

technical field [0001] The invention relates to the fields of genetic engineering and protein engineering, in particular to SUMO and SUMO protease genes, encoded proteins and applications thereof. Background technique [0002] The Escherichia coli expression system is often used to express recombinant proteins, but there are also some defects. When some foreign proteins are overexpressed, insoluble and non-functional inclusion body precipitation will often be formed [Villaverde A, Carrio M, M.2003. Proteinaggregation in recombinant bacteria : biological role of inclusionbodies.Biotechnol letters.25(17):1385-1395], fusion tag expression has been shown to be used to improve the yield and folding of foreign recombinant proteins [Esposito D, Chatterjee D, K.2006.Enhancement of soluble protein expression through the use of fusion tags. Curr. Opin. in Biotechnol. 17(4):353-358]. Traditional fusion tags include maltose-binding protein MBP, glutathione S-transferase GST, thioredoxi...

Claims

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Application Information

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IPC IPC(8): C12N15/30C12N15/57C07K14/395C12N9/60C12N15/70C12N15/66
CPCC07K14/395C07K2319/21C12N9/60C12N15/66C12N15/70C12N2800/101C12N2800/22
Inventor 范军周旋
Owner ANHUI AGRICULTURAL UNIVERSITY
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