Protein Production and Purification

The oxygen lability of [4Fe-4S] clusters requires that Fe-S enzymes be purified under strict anaerobic conditions.

An argon recirculated glove box operated at <0.2ppm O₂ was used for the anoxic purification of the RNA-dependent RNA polymerase of SARS-CoV-2.

Consumables required for any manipulations inside the chamber was purged of O₂ before use. Most plasticware was first autoclaved and then introduced into the chamber while hot and allowed to dry and equilibrate for at least 24h before use.

Plasticware that could not be autoclaved was allowed to equilibrate for a minimum of 1 week before use.

All buffers and solutions were transferred inside the chamber and allowed to equilibrate with the chamber’s atmosphere for at least 48h.

Expi293F cells at a final density of 3 × 10⁶ viable cells/mL (2.25 x 10⁹ cells at the time of transfection) were inoculated in 800 mL of fresh Expi293™ Expression Medium supplemented with 300 μM L-cysteine and 40 μM FeCl₃ or ⁵⁷FeCl₃ (for Mossbauer and EPR analyses) and transfected with the following combinations of constructs: 840 μg P3xFLAG-SARS-CoV-2-nsp12 alone (for Mossbauer and EPR studies) or 420 μg P3xFLAG-SARS-CoV-2-nsp12 and 840 μg of pIRES-nsp7/nsp8-Strep II (for the RdRp activity assays), which were combined with 48 ml of Opti-MEM™ I Reduced Serum Medium (ThermoFisher Scientific) and incubated for 5 min at room temperature. The plasmid DNA mix was then combined with a mix containing 2.6 ml ExpiFectamine™ 293 Reagent and 45 ml Opti-MEM™ I Reduced Serum Medium and incubated at room temperature for additional 20 min prior to addition to the cell suspension. 

Forty-eight hours after transfection, cells were spun down and washed twice with cold PBS prior to being taken inside the anaerobic chamber operated at <0.2ppm O₂ for lysis. Cells were lysed in 100 ml of lysis buffer (150 mM NaCl, 50 mM Na-HEPES pH 7.4, 10% (v/v) glycerol, 3 mM MgCl₂, 2 mM TCEP, 0.5% NP-40, EDTA-free protease inhibitor cocktail) and homogenized by gently pipetting up and down until no cell clumps were visible. 

After clearing up the insoluble fractions by centrifuging the homogenate at 40,000 x g for 30 min, 4 ml of pre-equilibrated FLAG-M2 beads were added to the lysate. Recombinant protein complexes were immunoprecipitated for 3 hours at room temperature, before being packed into a column. The resin was washed with 10 column volumes of lysis buffer, followed by 20 column volumes of lysis buffer supplemented with 300 mM NaCl. 

The FLAG-tagged nsp12 proteins or the 3xFLAG-nsp12/nsp7/nsp8-Strep tag II complexes were then eluted from the FLAG-M2 beads with 3xFLAG peptide dissolved in lysis buffer for 1 hour at room temperature. 

The eluates at ~50 μM were concentrated to ~250 μM using a 50 kDa molecular weight cut-off filter.