Fig. 1: Genome organization of SARS-CoV-2, purification of MPro^D48Y/ΔP168 dimer and autoprocessing of ⁽⁻¹⁰²⁾MPro^D48Y/ΔP168-GP-6H precursor.

A The ~30 kb genome codes for the various proteins in at least 12 open reading frames (ORFs). Two major polyproteins (pp) are encoded in ORFs, 1a (nsp1-nsp10) and 1ab (nsp11-nsp16), the processed proteins of which make up the replication/transcription complex. pp1ab is synthesized via a translation frameshifting (denoted FS) mechanism. The two virally encoded proteases PLPro (papain-like, black) and 3C-like main protease (MPro, red) are responsible for processing pp1a and pp1ab. In the precursor form, MPro is suggested to be anchored on either side with membrane-spanning helices (white bars) within nsp4 and nsp6. MPro is responsible for its own release (termed autoprocessing) and cleavage of the rest of the sites between nsp4 and nsp16. B Expression of MPro^D48Y/ΔP168-GP-6H. Uninduced (lane 1). Distribution of MPro^D48Y/ΔP168-GP-6H in the soluble (lane 2) and insoluble (lane 3) fractions quantified by subjecting cells (25 ml) to lysis followed by fractionation of the soluble and insoluble fractions and small-scale nickel-affinity chromatography (see “Methods” section for details). C Purified MPro^D48Y/ΔP168 dimer. D N-terminal autoprocessing of ⁽⁻¹⁰²⁾MPro^D48Y/ΔP168-GP-6H precursor (46.5 kDa) in E. coli results in MPro ^D48Y/ΔP168-GP-6H (34.7 kDa) and 11.8 kDa products. Only the 34.7 kDa product is retained in the column because of the 6H tag being at the C-terminus. The appearance of the 11.8 kDa upon cleavage was verified in an earlier work using a similar precursor construct (see Fig. 3a, c in ref. ⁶). Cells (12 ml) were harvested at the indicated time points, and equal volumes of the bound fractions following nickel-affinity chromatography (NAC) were analyzed by SDS-PAGE. Proteins were subjected to SDS-PAGE on 4–20% gradient gels. M denotes protein standards in kDa.