Improving soluble recombinant SARS-CoV-2 papain-like protease production in Escherichia coli through chaperonin and maltose-binding protein tag: purification and kinetic characterization.

Although COVID-19 is now becoming endemic, SARS-CoV-2 persists potential jeopardy to clinically vulnerable populations. Hence, further study is still necessary to discover novel antiviral agents against SARS-CoV-2 for proactive preparedness. SARS-CoV-2 papain-like protease (PL Pro) is a target enzyme for searching anti-Covid candidates. Our prior study revealed the major formation of inclusion bodies during PL Pro expression in E. coli RIPL. In this study, we tried using chaperonin in the E. coli Arctic Express system and both codon optimization and maltose-binding protein (MBP) fusion protein to make PL Pro more soluble. Recombinant PL Pro encoded on the pET21d(+) plasmid was expressed in E. coli Arctic express. However, the soluble protein yield remained low and unstable due to suboptimal codon usage in the insert gene. Whereas, fusion of the MBP protein with optimized codon of PL Pro enhanced the enzyme expression and solubility. Recombinant PL Pro cleaved the linker between MBP and PL Pro, which served as a cleavage site recognized by PL Pro (LKGG↓A). The purified enzyme from a 200-mL culture generated 1 mL of pure PL Pro enzyme at a 1.913 mg/mL concentration. It exhibited favorable activity against the Z-RLRGG-AMC substrate, with a Km value of 33.40 μM and a Vmax of 5.10 RFU/min.