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

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

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0045] The method for preparing a recombinant vector of pET28b-sumo(S) and pMF-ulp(S) of the present invention includes the following steps:

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

[0047] (2) Recombinant vectors of pET28b-sumo(S) and pMF-ulp(S) were constructed separately; the pMF-ulp(S) recombinant vector is selective to the vector. The present invention proves that ulp(S) is constructed in pET28b The vast majority of expression in vectors is precipitation of inclusion bodies, but soluble expression can be achieved in pMF vectors.

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

[0049] Using DNA recombination technology, insert the sumo(S) gene shown in SEQ ID NO:1 in the sequence table into the Nde Ⅰ and BamH Ⅰ of pET28b, and cut the ulp(S) gene and pMF ...

Example Embodiment

[0063] Example 1

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

[0065] The sumo(S) gene and pET28b vector were digested with Nde Ⅰ and BamH Ⅰ. The ulp(S) gene and pMF vector were digested with BamH Ⅰ and Hind Ⅲ. After the gel was recovered, the corresponding gene and vector were ligated with T4 DNA ligase at 16°C for 5 hours. The ligation product was transferred to E. coli DH5α competent cells. After incubation and refolding, it was coated on a resistant plate and cultured at 37°C for 12 hours. A single colony was picked and cultured in 5mL Luria-Bertani medium at 37°C and 220 rpm for 12 hours. The plasmid was extracted and sequenced after restriction enzyme digestion verification was correct.

[0066] Transform the correctly sequenced pET28b-sumo(S) and pMF-ulp(S) plasmids into BL21(DE3) competent cells, the control plasmids are pET28b-sumo(wt) and pET28b-ulp(wt), the control plasmids Transform into BL21(DE3) a...

Example Embodiment

[0072] Example 2

[0073] Construction and expression of ulp(S) recombinant vector containing six 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. After the plasmids were digested and verified correctly, these six tags The vector and ulp(S) were digested with BamH Ⅰ and Hind Ⅲ and ligated. After successful transformation and digestion, the six plasmids were transferred to BL21(DE3) competent cells. A single colony was picked from the plate and placed in 5 mL Luria. -Bertani medium 37℃, 220rpm shaking culture, the bacteria grow to OD 600 When it reached 0.6, IPTG with a final concentration of 0.5mmol / L was induced at 28°C for 12h, and then the cells were collected and ultrasonically broken. After freezing and centrifugation, the superalbumin samples were subjected to SDS-PAGE electrophoresis. Such as image 3 Shown. The results showed that the expressi...

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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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