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

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

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_3CL(pro)) 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_3CL(pro), in this report the synthetic genes encoding 3CL(pro) 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-Ni(2+) affinity chromatography and FPLC system, and its sequence was determined by LC/MS with the residue coverage of 46.4%.