Plasmid construction and protein expression

The DNA encoding the middle domain of human Paip1 (Paip1M, residues 78-296) was amplified by polymerase chain reaction (PCR) and was subsequently inserted to pET28a-sumo plasmid between the restriction enzyme sites BamHI and XhoI according to Gao et al.⁹. The genes encoding SARS-CoV SUD-core (nsp3 389-652) and SARS-CoV-2 SUD-core (nsp3 residues 413–676) were synthesized with optimized codon for E. coli (Sangon Biotech) and cloned into the pET-Duet-1 vector between the restriction BamHI and HindIII, which expressed the N-terminal 6× His-tag fused SUD-core. The plasmid encoding mutant SARS-CoV-2 SUD-core-CC containing L516C and Y647C and other 17 mutant plasmids as shown in Fig. 3 and Supplementary Fig. ⁷ were generated by site-directed mutagenesis (QuikChange™). All constructures were verified by DNA sequencing.

The expression of SUD-core proteins and Paip1M follows the similar protocol. Briefly, plasmids were transferred to C3016H competent cells (New England Biolabs). A single colony was picked to inoculate lysogeny broth (LB) medium at 37 °C till a density of OD600 ~ 1.0. Isopropyl-D-1-thiogalactopyranoside (IPTG) was then added to the culture (final concentration ~0.5 mM) to induce the expression and the culturing continued for another 20 h at 18 °C. The bacteria culture was harvested by centrifugation at 2991 × g and stored at −80 °C before use.