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Expression and purification method of coronavirus main protease

A main protease, expression and purification technology, applied in the field of protein purification, can solve the problems of cumbersome steps and low yield, and achieve the effect of solving the problems of low yield and cumbersome steps

Pending Publication Date: 2021-10-12
SHENZHEN INST OF ADVANCED TECH
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  • Description
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Problems solved by technology

[0004] The object of the present invention provides a new expression and purification method of natural coronavirus main protease, to solve the problems of low yield and cumbersome steps in existing methods

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  • Expression and purification method of coronavirus main protease
  • Expression and purification method of coronavirus main protease
  • Expression and purification method of coronavirus main protease

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Embodiment 1

[0067] The whole gene encoding SARS-CoV-2 Mpro was synthesized and codon optimized for expression in Escherichia coli.

[0068] Construction of His-SUMO (COV) plasmid: The SARS-CoV-2 Mpro gene connected with silk amino acid is directly connected after the SUMO gene on the pET 28b-SUMO plasmid backbone. The purpose of the design is to use SUMO protease (Ulp1) to recognize the tertiary structure of the SUMO-recombinant protein and cut it to produce the natural main protease.

[0069] Specifically: a. Use the pET 28b-SUMO plasmid as a template, design upstream and downstream primers, and perform PCR amplification. The obtained PCR product is purified and treated with Dpn-I, and used as the backbone of the new plasmid.

[0070] b. Using the 2019-nCoV-pET 30a(+) plasmid as a template, design upstream and downstream primers (the 5' ends of the upstream and downstream primers have homologous sequences at both ends of the backbone) for PCR amplification, and the resulting PCR products...

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Abstract

The invention provides an expression and purification method of natural coronavirus main protease. The expression and purification method comprises the following steps of carrying out plasmid recombination, induced protein expression and purification on the natural main protease to generate fusion protein, and carrying out purification, enzyme digestion and separation on the fusion protein to obtain the natural main protease without redundant residues at the N-terminal and the C-terminal. By adopting the method, the problems of tedious experimental steps and low yield in the existing traditional method can be solved.

Description

technical field [0001] The invention relates to the technical field of protein purification, in particular to a method for expressing and purifying coronavirus protease. Background technique [0002] Coronavirus was first isolated from chickens in 1937 (Cunningham and Stuart, 1947), and in 1967 (Estola and Weckstrom, 1967), it was seen under the electron microscope that there were spikes on the virus envelope, and the shape of the virus particles was similar to that of European emperors. The crown structure of the virus is similar, so it is called a coronavirus. Human coronaviruses were first isolated and identified more than 60 years ago from patients with respiratory infections, from which two distinct viruses 229E (Hamre and Procknow, 1966) and OC43 (McIntosh et al., 1967) were initially identified . Because it was difficult to find a cell culture method suitable for culturing all types of coronaviruses in the early days, many early epidemiological studies were serologi...

Claims

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Application Information

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IPC IPC(8): C12N9/50C12N15/70C12N15/62
CPCC12N9/506C12N15/70C12N2800/22C07K2319/21C07K2319/95
Inventor 李楠王维杰王蕾
Owner SHENZHEN INST OF ADVANCED TECH
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