Production of a functionally active recombinant SARS-CoV-2 (COVID-19) 3C-Like protease and a soluble inactive 3C-like protease-RBD chimeric in a prokaryotic expression system

Abstract

During the SARS-CoV-2 intracellular life-cycle, two large polyproteins, pp1a and pp1ab, are 29 produced. Processing of these by viral cysteine proteases, the papain-like protease (PLpro) and 30 the chymotrypsin-like 3C-like protease (3CL-pro) release non-structural proteins necessary for 31 the establishment of the viral replication and transcription complex (RTC), crucial for viral 32 replication. Hence, these proteases are considered prime targets against which anti-COVID-19 33 drugs could be developed. Here, we describe the expression of a highly soluble and functionally 34 active recombinant 3CL-pro using Escherichia coli BL21 cells. In addition, we assessed the 35 ability of our 3CL-pro to function as a carrier for the Receptor Binding Domain (RBD) of the 36 Spike protein. The co-expressed chimeric protein, 3CLpro-RBD, did not exhibit 3CL-pro 37 activity, but its enhanced solubility made purification easier and improved RBD antigenicity 38 when tested against serum from vaccinated individuals in ELISAs.