Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 32, Issue 2, Pages 302-308Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2003.08.016
Keywords
Severe Acute Respiratory Syndrome (SARS); SARS coronavirus; 3C like protease; molecular cloning; expression; purification; mass spectrometric characterization
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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 (similar to15 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%. (C) 2003 Elsevier Inc. All rights reserved.
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