Profiling of Substrate Specificities of 3C-Like Proteases from Group 1, 2a, 2b, and 3 Coronaviruses

Chuck, Chi-Pang; Chow, Hak-Fun; Wan, David Chi-Cheong; Wong, Kam-Bo

Cloning, expression and purification of SARS-CoV 3CL pro were described previously [26] . Codon-optimized DNA sequences encoding HCoV-NL63 (GenBank AY567487) and HCoV-OC43 (GenBank AAX85666), and IBV (GenBank M95169) 3CL pro were purchased from Mr. Gene (http://mrgene.com). The coding sequences of 3CL pro from HCoV-NL63, HCoV-OC43 and IBV were sub-cloned and expressed in E. coli strain BL21 (DE3) pLysS as fusion proteins with N-terminal tags of poly-histidine-small ubiquitin-related modifier (His 6 -SUMO) or poly-histidine-maltose binding protein (His 6 -MBP). Protein expression was induced by addition of 0.1 mM of isopropyl b-D-1-thiogalactopyranoside. After overnight incubation at 25uC, cells were harvested by centrifugation and resuspended in buffer A (20 mM Tris, pH 7.8, 150 mM NaCl and 1 mM tris(2-carboxyethyl)phosphine) with 30 mM imidazole and disrupted by sonication. Soluble fraction was subject to immobilized metal ion affinity chromatography for purification as described for SARS-CoV 3CL pro [26] . The His 6 -SUMO or His 6 -MBP tags were removed by protease digestion using sentrin-specific protease 1 or factor Xa, respectively, followed by immobilized metal ion affinity chromatography. Native 3CL pro were finally purified by G75 size exclusion column and stored in buffer A. Elution profiles of size exclusion chromatography indicated that all 3CL pro purified were dimeric.