Recombinant SARS-CoV-2 3CLpro Purification

Gene encoding recombinant 3CLpro from SARS‐CoV‐2 was introduced into pET28b(+) bacterial expression vector by GenScript Inc (Piscataway, NJ), and the His × 6‐tagged 3CLpro protein was expressed and purified as described previously.⁴¹ (link) Briefly, recombinant 3CLpro was expressed in Escherichia coli BL21(DE3)CodonPlus‐RIL cells (Agilent Technologies, Santa Clara, CA) grown in terrific broth. The cell lysate was loaded onto a ProBond nickel‐chelating resin (Thermo Fisher Scientific), followed by a HiLoad Superdex 200 size‐exclusion column (GE Healthcare), using an AKTA purifier core system (GE Healthcare). The final protein sample was purified in a buffer containing 20 mM Hepes, pH 7.5, 150 mM NaCl, and 0.5 mM tris(2‐chloroethyl) phosphate (TCEP). The final protein sample was collected and concentrated to approximately 150 µM, as determined by Bradford assay (Bio‐Rad Laboratories). The sample purity was assessed by SDS‐PAGE.