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Exogenous recombinant expression and purification method for crystallizable mature SARS coronavirus non-structural protein 12 (sars-nsp12)

A protein and fusion protein technology, applied in the field of RNA polymerase nsp12 preparation, can solve the problems of low yield of target protein, low biological activity of recombinant expressed protein, unstable physical and chemical properties of recombinant protein, etc., and achieve stable physical, chemical and biological stability Effect

Inactive Publication Date: 2013-07-03
TIANJIN INT JOINT ACADEMY OF BIOTECH & MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims to solve many important problems and defects caused by the recombinant expression of the SARS coronavirus RNA-dependent RNA polymerase nsp12 in the existing technical solutions, especially: low yield of the target protein, low biological activity of the recombinantly expressed protein, and low physical and chemical bioactivity of the recombinant protein. The problem and defect that character is unstable and very easy to degrade, provide the RNA-dependent RNA polymerase (RNA-dependent RNA polymerase, RdRP)-nonstructural protein No. 12 (nsp12) that SARS coronavirus has normal biological activity Methods of type cloning, recombinant expression, purification and crystallization

Method used

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  • Exogenous recombinant expression and purification method for crystallizable mature SARS coronavirus non-structural protein 12 (sars-nsp12)
  • Exogenous recombinant expression and purification method for crystallizable mature SARS coronavirus non-structural protein 12 (sars-nsp12)
  • Exogenous recombinant expression and purification method for crystallizable mature SARS coronavirus non-structural protein 12 (sars-nsp12)

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Embodiment 1

[0031] Cloning of the wild-type gene of the RNA polymerase (RNA-dependent RNA polymerase, RdRP)-nonstructural protein No. 12 (nsp12) of embodiment 1 SARS coronavirus RNA and the construction of expression plasmid

[0032] The small ubiquitin-like modifier protein gene (Saccharomyces cerevisiae S288c Smt3p (SMT3) mRNA 1-291) was cloned into the pET-26b vector (from Novagen, Cat. No. 69862-3), a pET-26b-S-CHis6 construct was formed. Subsequently, the nsp 12 gene (SARS coronavirus BJ01, complete genome positions 13111-15903) was cloned into the pET-26b-S-CHis6 plasmid with BamHI and XhoI as the 5' end and 3' end restriction sites, respectively, to form pET-26b - Construction of the S-nsp12-CHis6 plasmid.

Embodiment 2

[0033] Example 2 Construction of an expression plasmid for excising redundant amino acid residues at the N-terminal of nsp12 to form a wild-type protease

[0034] The ubiquitin-like protein processing enzyme (Saccharomyces cerevisiae S288c Ulp1p (ULP1) mRNA 1207-1866) was cloned into the pET11a vector with NdeI and BamHI as the 5' end and 3' end restriction sites respectively by molecular cloning technology to form pET- Construction of 11a-Ulp expression plasmid.

Embodiment 3

[0035] Example 3 Expression of wild type N-terminal nsp12 protein in prokaryotic system

[0036] The expression plasmids pET-26b-S-nsp12-CHis6 and pET-11a-Ulp formed in Example 1 and Example 2 were transformed into Escherichia coli BL21(DE3)pLysS, and screened with ampicillin and kanamycin Obtain a host strain carrying both expression plasmids. Cultivate the obtained host strain at 37 degrees Celsius to OD600>0.7, add IPTG to a final concentration of 200mM and culture at 20 degrees Celsius for 16 hours, during which the induced expression of ubiquitin-like protein processing enzymes will automatically convert smt3-nsp12 Protein cleavage processes to form nsp12 protein with wild-type nitrogen terminus (SADAS). The cells were collected by centrifugation and resuspended with phosphate buffered saline, centrifuged again to collect plaques and refrigerated at minus 20°C.

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Abstract

According to the invention, a small ubiquitin-like modifier protein gene and a nsp12 gene are connected into a pET-26b vector to realize formation of a pET-26b-S-nsp12-CHis6 expression plasmid, a ubiquitin-like protein processing enzyme is linked into a pET11a vector to realize formation of a pET-11a-Ulp expression plasmid, and the aforementioned pET-26b-S-nsp12-CHis6 and the pET11a-Ulp expression plasmid are transformed into an escherichia coli BL21(DE3)pLysS to realize expression and purification of wild type protein of the nsp12. The obtained wild type protein of the nsp12 has normal RNA polymerase biological activity that should own by the wild-type protein, and can be used for crystallization experiments after necessary purification treatment, and can form a protein crystal. A method for exogenous recombinant expression and purification of crystallizable mature SARS coronavirus non-structural protein 12 (sars-nsp12) is provided by the invention.

Description

technical field [0001] The invention relates to a recombinant polymerase, in particular to a method for preparing recombinant RNA-dependent RNA polymerase nsp12 with a wild nitrogen terminal, and belongs to the field of recombinant polymerases. Background technique [0002] Coronavirus (Coronaviridae / Coronavirus, CoV) is a class of pathogenic microorganisms that are harmful to humans and livestock. The global outbreak of SARS-CoV in 2003 has caused serious threats and major losses to human health and the global economy. . Coronavirus has the largest known positive-sense RNA genome. After infecting cells, it obtains a series of viral proteins through direct translation and discontinuous transcription-translation, including non-structural proteins, structural proteins and accessory proteins. For non-structural proteins and accessory proteins that play an important role in the transcription / replication process of coronaviruses, due to their low similarity to the protein seque...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/63C12N1/21C12N15/70C12N9/12C12R1/19
Inventor 饶子和杨诚娄智勇孙玉娜王权李洋刘素晓李彩霞王静陈卫强
Owner TIANJIN INT JOINT ACADEMY OF BIOTECH & MEDICINE
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