Bl21 codonplus cells

The kanamycin-resistant pET29a(+) expression plasmid encoding for N-terminally tagged mouse (Mus musculus) ATE1-1 (Uniprot ID Q9Z2A5) was prepared as previously described^{33 (link)}. The expression plasmid was transformed into chemically competent BL21 Codon Plus cells (Agilent part # 230245), spread onto LB agar plates supplemented with 100 μg/mL kanamycin and 50 μg/mL chloramphenicol and grown overnight at 37 °C. Expression of MmATE1-1 was identical to that of ScATE1 (vide supra) and cells were grown in the presence of supplemented L-Cys and ferric citrate, as described (vide supra), except that the antibiotics kanamycin and chloramphenicol were supplemented to 100 and 50 μg/mL (final concentrations), respectively, instead of ampicillin. The purification of MmATE1-1 was identical to that of ScATE1 (vide supra).

The coding sequences for the VL and VH regions of selected Fabs were cloned into a custom expression vector with protein expression driven by a ptac promoter, with a C-terminal 6xHis tag at the end of CH1 domain on heavy chain. Fab proteins were expressed in E. coli. BL21 Codon Plus cells (Agilent, Santa Clara, CA, USA). Actively growing cells (O.D. 0.8) were induced using 0.4 mM IPTG and incubated at 24 °C for 12 h. Cells were lysed and the fab proteins were purified from clarified lysate using Ni-NTA Sepharose resin (GE Healthcare) using manufacturer recommended protocols.

A plasmid (pETM33_NSP5_M^pro; a gift from Ylva Ivarsson (Addgene plasmid # 156475; http://n2t.net/addgene:156475; RRID: Addgene_156475)) encoding the SARS-CoV-2 M^pro was transformed into Escherichia coli (E. coli) BL21-CodonPlus cells (Agilent Technologies). The protein was expressed upon induction with 0.1 mM isopropyl‐β‐D‐thiogalactopyranoside (IPTG) at 37 °C for 2.50 h. The cells were harvested by centrifugation (4000 g, 10 min, 4 °C) and the cell pellet was resuspended in 10 mL lysis buffer containing 50 mM Tris (pH 8), 300 mM NaCl, 10 mM β-mercaptoethanol (β-ME), 1 mM PMSF and 10 % (v/v) glycerol and lysed by sonication [21] , [22] , [26] , [27] (link). The supernatant was collected after centrifugation (18000 g, 90 min, 4 °C) and incubated with pre-equilibrated glutathione (GSH) beads at 4 °C for 2 h. The beads were washed with a buffer containing 50 mM Tris (pH 7), 150 mM NaCl, 10 mM β-ME, 1 mM EDTA, 0.01 % Triton X-100 % and 10 % glycerol. Following washes, the beads were incubated with the PreScission Protease (GE Healthcare #27–0843–01) in a buffer containing 50 mM Tris (pH 7), 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.01 % Triton X-100 % and 10 % glycerol) at 4 °C for 16 h to cleave M^pro from the GST-tag. The supernatant containing SARS-CoV-2 M^pro protease was collected after centrifugation at 500 g at 4 °C for 10 min.