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.

De Marco Verissimo, Carolina ORCID: 0000-0003-1762-6387, López Corrales, Jesús ORCID: 0000-0003-2996-7904, Dorey, Amber L, Cwiklinski, Krystyna ORCID: 0000-0001-5577-2735, Lalor, Richard, Calvani, Nichola Eliza Davies ORCID: 0000-0002-8787-6755, Jewhurst, Heather L, Flaus, Andrew, Doyle, Sean ORCID: 0000-0003-1679-3247 and Dalton, John P (2022) 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. Epidemiology and infection, 150. e128-.

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Official URL: http://dx.doi.org/10.1017/s0950268822001078

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 <i>Escherichia coli</i> 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.

Item Type:	Article
Uncontrolled Keywords:	Humans, Peptide Hydrolases, Cysteine Endopeptidases, Antiviral Agents, COVID-19, SARS-CoV-2, COVID-19 Vaccines, Coronavirus 3C Proteases
Divisions:	Faculty of Health and Life Sciences Faculty of Health and Life Sciences > Institute of Infection, Veterinary and Ecological Sciences
Depositing User:	Symplectic Admin
Date Deposited:	23 Jun 2022 07:23
Last Modified:	14 Mar 2024 17:46
DOI:	10.1017/s0950268822001078
Open Access URL:	https://www.cambridge.org/core/journals/epidemiolo...
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3156986