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Efficient soluble expression method of active recombinant ribonuclease inhibitor

A technique for ribonuclease and inhibitors, which is applied in the field of prokaryotic expression of recombinant ribonuclease inhibitors, and can solve problems such as no reports

Inactive Publication Date: 2011-11-09
WEIZHI LANSI MEDICAL TECH BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although commercially available recombinant mouse RI has been provided, and the complete gene sequence encoding mouse RI has been reported (NCBI sequence number: AF071546; NM_145135), its effective recombinant expression method for active products in Escherichia coli or other expression hosts , no report yet

Method used

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  • Efficient soluble expression method of active recombinant ribonuclease inhibitor
  • Efficient soluble expression method of active recombinant ribonuclease inhibitor
  • Efficient soluble expression method of active recombinant ribonuclease inhibitor

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Embodiment Construction

[0024] The statements of the following embodiments are for better understanding of the principle and application of the present invention, but not to limit the application scope of the present invention.

[0025] 1. Materials and methods:

[0026] Bovine pancreas RNase A was purchased from Beijing Qinyuan Huizhi Biotechnology Co., Ltd.; hRI (recombinant human RI) was a product of Promega (trade name RNasin Ribonuclease Inhibitor), and yeast rRNA was purchased from Shanghai Kaiyang Biotechnology Co., Ltd. and purified before use deal with. Ferulic acid (FA) was purchased from Sinopharm Chemical Reagent Co., Ltd. Ni-IDAAgarose was purchased from Weiglass Biotechnology Co., Ltd.

[0027] Quantification of purified protein: use 5 μl, 10 μl, and 20 μl of bovine serum albumin (BSA) at a concentration of 0.2 mg / ml as a standard control, and perform SDS-PAGE electrophoresis together with the purified protein. Coomassie brilliant blue staining, QuantiScan software to scan the gel in...

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Abstract

The invention provides an efficient soluble expression method of an active recombinant ribonuclease inhibitor, belonging to the technical field of biological engineering. The invention discloses an effective method for obtaining an active recombinant ribonuclease inhibitor, which is simple and easy to implement and has low cost. A recombinant plasmid vector of a coding gene for expressing a mouse ribonuclease inhibitor (mRI) is converted into engineering bacteria with protease gene defect phenotypes, and soluble expression of higher-level active products can be obtained by appropriate induction.

Description

Technical field: [0001] The invention belongs to the field of bioengineering, in particular to the prokaryotic expression of recombinant ribonuclease inhibitors. Background technique: [0002] Ribonuclease inhibitor (RNase Inhibitor, RI) is a protein molecule with a molecular weight of about 50KDa, which can bind and inhibit various members of the mammalian pancreatic ribonuclease (RNase) superfamily. [0003] RI is a cytoplasmic protein that is ubiquitous in many mammalian tissues. It binds tightly with various ribonucleases to form a 1:1 complex, inhibiting the activity of these ribonucleases. Although the homology of some members of the RNase superfamily is very different, the complexes formed by RI and them have dissociation constants at the level of 0.5-200fM (Lee FS, Shapiro R, Vallee BL. Tight-binding inhibition ofangiogenin and ribonuclease A by placental ribonuclease inhibitor. Biochemistry, 1989, 28: 225-30; Shapiro R, Vallee BL. Interaction of human placental ri...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/70C07K14/47
Inventor 王楠孙婧慧葛莹
Owner WEIZHI LANSI MEDICAL TECH BEIJING
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