Expression and Purification of CoV-2 3CL Protease
The expression and purification strategy followed previous studies on SARS CoV-1 3CLpro.58 Genes encoding SARS CoV-1 3CLpro and SARS CoV-2 Mpro MN908947.3 were synthesized with codon usage optimized for Escherichia coli expression (Genscript and IDT). Genes were cloned into a modified pET24a vector to produce a TEV-cleavable N-terminal 6xHis tag under the T7 promoter control. BL21(DE3) cells (LifeTech) were grown in Terrific Broth (Teknova) at 37 °C until an OD600 = 0.6–0.8 and induced with 0.4 M IPTG for 5 h at 30 °C. Cell pellets were stored at −80 °C until purification. Cell pellets were resuspended and lysed by microfluidization in Buffer A (20 mM Tris pH 8.0 + 150 mM NaCl). Lysates were clarified at 25,000 × g for 1 h at 4 °C. The soluble lysate was loaded onto a nickel affinity column (Probond) and washed sequentially with Buffer A + 5 mM and 20 mM imidazole. The proteases were eluted with Buffer A + 300 mM imidazole. The His-tag was removed by TEV protease (1:40) during an overnight dialysis step in Buffer A + 1 mM DTT. The CoV Mpro proteins were further purified on a HiTrap Q hp (GE Healthcare), and the Q-flow-through material was concentrated and loaded onto a 26/60 Superdex-75 (GE Healthcare) gel filtration column equilibrated with Buffer B (20 mM Tris pH 7.8 + 150 mM NaCl + 1 mM EDTA + 1 mM DTT). Final proteins were concentrated from 5 to 25 mg/mL and were directly moved to crystallization experiments or snap-frozen in liquid nitrogen for storage.