Over-expression and Purification of Recombinant 3C-like Protease from Black Queen Cell Virus in Escherichia coli

Abstract

Black queen cell virus is one of the members belonging to Picornavirus, which possesses a 3C-like protease. Viral replication and capsid assembly in the order Picornavirales require polyprotein proteolysis processing, which is performed by 3C or 3C-like (3CL) proteases. Therefore, viral proteases are attractive targets for anti-viral therapy. The complete 3C-like protease gene from Black queen cell virus was cloned into pGEM-3Zf(+) vector, and the amino acid sequence revealed a high homology (99.1%) to 3C-like protease deposited in a reference Genbank EF517515.1. For the expression, 3C-like protease gene was subcloned into pMAL-C2 vector system. The expression level of MBP-BQCV-3CL protein was evaluated under different standards of cell optical density, IPTG concentration, induction time, and induction temperature. As the result, the optimal condition was confirmed as the induction with 0.1mM IPTG at 25°C for 6 hours, and a purification step using amylose resin reached a high protein yield of approximately 2.22mg per 1ml of cultured cells. Purified MBP-BQCV-3CL proteins can provide an important antigen source to generate monoclonal antibody and will become a potential candidate for anti-viral drugs, which can inactivate 3C-like protease function as well as BQCV replication.