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Molecular cloning, expression, purification, and mass spectrometric characterization of 3C-like protease of SARS coronavirus

2003; Elsevier BV; Volume: 32; Issue: 2 Linguagem: Inglês

10.1016/j.pep.2003.08.016

1096-0279

Haifang Sun, Hai‐Bin Luo, Changying Yu, Tao Sun, Jing Chen, Shuying Peng, Jun Qin, Jianhua Shen, Yiming Yang, Youhua Xie, Kaixian Chen, Yuan Wang, Xu Shen, Hualiang Jiang,

Viral gastroenteritis research and epidemiology

Severe acute respiratory syndrome (SARS) is an acute respiratory illness, which has broken out in China. It has been known that SARS coronavirus (SARS_CoV) is a novel human coronavirus and is responsible for SARS infection. Belonging to one of the major proteins associated with SARS_CoV, SARS 3C-like protease (SARS_3CLpro) functions as a cysteine protease engaging in the proteolytic cleavage of the viral precursor polyprotein to a series of functional proteins required for coronavirus replication and is considered as an appealing target for designing anti-SARS agents. To facilitate the studies regarding the functions and structures of SARS_3CLpro, in this report the synthetic genes encoding 3CLpro of SARS_CoV were assembled, and the plasmid was constructed using pQE30 as vector and expressed in Escherichia coli M15 cells. The highly yielded (∼15 mg/L) expressed protease was purified by use of NTA-Ni2+ affinity chromatography and FPLC system, and its sequence was determined by LC/MS with the residue coverage of 46.4%.