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.
Keywords: 3C-Like protease; COVID-19; Chimer; SARS-CoV-2; main protease.
【저자키워드】 COVID-19, SARS-CoV-2, 3C-like protease, main protease., Chimer, 【초록키워드】 coronavirus, Transcription, drug, protease, severe acute respiratory syndrome Coronavirus, inhibitors, Spike protein, non-structural proteins, Receptor binding domain, Papain-like protease, Protein, serum, COVID-19 vaccines, cells, RBD, viral replication, non-structural protein, target, Proteases, antigenicity, pp1a, PLPro, disease, expression, ELISAs, function, Escherichia coli, chimeric, cysteine, acute respiratory syndrome, Serine, acute respiratory syndrome coronavirus, acute respiratory syndrome coronavirus 2, enzyme, cysteine proteases, complex, cysteine protease, processing, chymotrypsin, Papain, vaccinated individuals, purification, vaccinated individual, carrier, solubility, inhibitory, polyproteins, pp1ab, approach, produced, tested, blocked, addition, RTC, exhibit, co-expressed, dimeric, the Spike, Escherichia coli BL21, 【제목키워드】 Production, chimeric, inactive, prokaryotic expression system,