The Mpro coding sequence was codon optimised for expression in E. coli and synthesised by Integrated DNA technologies (IDT). The Mpro expression construct used for crystallization comprises an N-terminal GST region, an Mpro autocleavage site, the Mpro coding sequence, a hybrid cleavage site recognizable by 3C HRV protease and a C-terminal 6-Histidine tag54 (link). The overall construct was flanked by In-Fusion compatible ends for insertion into BamHI-XhoI cleaved pGEX-6P-1 (Sigma). An additional Mpro construct was generated with an extended 10-Histidine tag, for enhanced binding to the sensor surface in SPR assays. This construct was amplified by PCR from the above version, with the C-terminal primer incorporating a further 4-Histidines. The resulting amplicon was then inserted into BamHI-XhoI cleaved pGEX-6P-1 by In-Fusion cloning.
The PLpro expression construct was similarly optimised and synthesised and comprised an N-terminal 10 Histidine tag followed by the PLpro sequence (Nsp3 region E746-K1060). This was then directly inserted into NcoI-HindIII digested pOPINF via In-Fusion compatible ends. pOPINF was a gift from Ray Owens (University of Oxford)55 (link) (Addgene plasmid # 26042 ;http://n2t.net/addgene:26042 ; RRID:Addgene_26042).
The PLpro expression construct was similarly optimised and synthesised and comprised an N-terminal 10 Histidine tag followed by the PLpro sequence (Nsp3 region E746-K1060). This was then directly inserted into NcoI-HindIII digested pOPINF via In-Fusion compatible ends. pOPINF was a gift from Ray Owens (University of Oxford)55 (link) (Addgene plasmid # 26042 ;
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