Optimization of expression conditions and determination the proteolytic activity of codon-optimized SARS-CoV-2 main protease in Escherichia coli.

Yunyu CHEN; Zhenghao FU; Gangan YAN; Yuan LIN; Xiaoping LIU

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Optimization of expression conditions and determination the proteolytic activity of codon-optimized SARS-CoV-2 main protease in Escherichia coli.

Author: Yunyu CHEN ¹ ; Zhenghao FU ¹ ; Gangan YAN ¹ ; Yuan LIN ² ; Xiaoping LIU ¹
Author Information

1. Institute for Drug Screening and Evaluation, Wannan Medical College, Wuhu 241002, Anhui, China.
2. State Key Laboratory of Bioactive Substances and Function of Natural Medicine, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Publication Type:Journal Article
Keywords: codons optimization; fluorescence resonance energy transfer (FRET); main protease; prokaryotic expression; severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
MeSH: COVID-19; Codon/genetics*; Cysteine Endopeptidases/genetics*; Escherichia coli/genetics*; Humans; Peptide Hydrolases; SARS-CoV-2; Viral Nonstructural Proteins/genetics*
From: Chinese Journal of Biotechnology 2021;37(4):1334-1345
CountryChina
Language:Chinese
Abstract: The main protease (Mpro) of SARS-CoV-2 is a highly conserved and mutation-resistant coronaviral enzyme, which plays a pivotal role in viral replication, making it an ideal target for the development of novel broad-spectrum anti-coronaviral drugs. In this study, a codon-optimized Mpro gene was cloned into pET-21a and pET-28a expression vectors. The recombinant plasmids were transformed into E. coli Rosetta(DE3) competent cells and the expression conditions were optimized. The highly expressed recombinant proteins, Mpro and Mpro-28, were purified by HisTrapTM chelating column and its proteolytic activity was determined by a fluorescence resonance energy transfer (FRET) assay. The FRET assay showed that Mpro exhibits a desirable proteolytic activity (25 000 U/mg), with Km and kcat values of 11.68 μmol/L and 0.037/s, respectively. The specific activity of Mpro is 25 times that of Mpro-28, a fusion protein carrying a polyhistidine tag at the N and C termini, indicating additional residues at the N terminus of Mpro, but not at the C terminus, are detrimental to its proteolytic activity. The preparation of active SARS-CoV-2 Mpro through codon-optimization strategy might facilitate the development of the rapid screening assays for the discovery of broad-spectrum anti-coronaviral drugs targeting Mpro.