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. (20th June 2022)

• Record Type: Journal Article
• Authors: De Marco Verissimo, Carolina; López Corrales, Jesús; Dorey, Amber L.; Cwiklinski, Krystyna; Lalor, Richard; Calvani, Nichola Eliza Davies; Jewhurst, Heather L.; Flaus, Andrew; Doyle, Sean; Dalton, John P.
• Is Part Of: Epidemiology and infection. Volume 150(2022)
• Publication Date: 2022-06-20
• Subjects: 3C-Like protease -- Chimer -- COVID-19 -- main protease -- SARS-CoV-2; Communicable diseases -- Periodicals; Epidemiology -- Periodicals; 614.4
• DOI: 10.1017/S0950268822001078
• Languages: English
• ISSNs: 0950-2688

Abstract

Abstract: During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) intracellular life-cycle, two large polyproteins, pp1a and pp1ab, are produced. Processing of these by viral cysteine proteases, the papain-like protease (PLpro) and the chymotrypsin-like 3C-like protease (3CL-pro) release non-structural proteins necessary for the establishment of the viral replication and transcription complex (RTC), crucial for viral replication. Hence, these proteases are considered prime targets against which anti-coronavirus disease 2019 (COVID-19) drugs could be developed. Here, we describe the expression of a highly soluble and functionally active recombinant 3CL-pro using Escherichia coli BL21 cells. We show that the enzyme functions in a dimeric form and exhibits an unexpected inhibitory profile because its activity is potently blocked by serine rather than cysteine protease inhibitors. In addition, we assessed the ability of our 3CL-pro to function as a carrier for the receptor binding domain (RBD) of the Spike protein. The co-expressed chimeric protein, 3CLpro-RBD, did not exhibit 3CL-pro activity, but its enhanced solubility made purification easier and improved RBD antigenicity when tested against serum from vaccinated individuals in ELISAs. Chimeric proteins containing the 3CL-pro could represent an innovative approach to developing new COVID-19 vaccines.