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    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 and López Corrales, Jesús and Dorey, Amber L. and Cwiklinski, Krystyna and Lalor, Richard and Calvani, Nichola Eliza Davies and Jewhurst, Heather L. and Flaus, Andrew and Doyle, Sean 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. ISSN 0950-2688

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    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.

    Item Type: Article
    Keywords: 3C-Like protease; Chimer; COVID-19; main protease; SARS-CoV-2;
    Academic Unit: Faculty of Science and Engineering > Biology
    Item ID: 17037
    Identification Number: https://doi.org/10.1017/S0950268822001078
    Depositing User: Dr. Sean Doyle
    Date Deposited: 20 Mar 2023 11:51
    Journal or Publication Title: Epidemiology and Infection
    Publisher: Cambridge University Press
    Refereed: Yes
    URI:
    Use Licence: This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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