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, C.; Lopez-Corrales, J.; Dorey, A. L.; Cwiklinski, K.; Lalor, R.; Calvani, N. E. D.; Jewhurst, H. L.; Doyle, S.; Dalton, J. P.

doi:10.1101/2022.03.25.485815

bioRxiv · 10.1101/2022.03.25.485815

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, C.·Lopez-Corrales, J.·Dorey, A. L.·Cwiklinski, K.·Lalor, R.·Calvani, N. E. D.·Jewhurst, H. L.·Doyle, S.·Dalton, J. P.

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Abstract

During the 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-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. 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. When used to immunise mice, the 3CLpro-RBD chimer elicited antibodies mainly to the 3CL-pro portion of the molecule indicating that a different chimeric composition (i.e., RBD/full Spike-3CLpro) or expression system (i.e., mammalian cells), might be required to produce and deliver a RBD with immunogenicity similar to the native protein. Chimeric proteins containing the 3CL-pro could represent an innovative approach to developing new COVID-19 vaccines.

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De Marco Verissimo, C., Lopez-Corrales, J., Dorey, A. L., Cwiklinski, K., Lalor, R., Calvani, N. E. D., Jewhurst, H. L., Doyle, S., Dalton, J. P.. 2022-03-25. 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. https://doi.org/10.1101/2022.03.25.485815

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