Superose 12 column

The molecular mass of ArsR protein ArsR1 or ArsR2 was measured using an ÄKTA Pure fast protein liquid chromatography system (Cytiva, Marlborough, MA, USA). First, 500 μL of 40 μM ArsR1 or 37 μM ArsR2 protein in 10 mM MOPS/0.1 M NaCl/15% glycerol buffer (pH 7.5) was loaded onto a Superose 12 column (Cytiva, Marlborough, MA, USA) pre-equilibrated by the same buffer at a flow rate 0.5 mL min⁻¹ and elution was monitored at 280 nm. The calibration was carried out with gel filtration standards ribonuclease A, chymotrypsinogen A, ovalbumin, and albumin in a concentration of 1 mg mL⁻¹. Then, 100 μM sodium arsenite or 1 mM diamide was mixed with the proteins and incubated for 10 min at 30 °C to investigate how these compounds influence ArsR oligomeric state.

Plasmids were transformed into BL21-DE3 cells (Agilent) and induced for expression at 0.7–0.8 optical density with 1 mM isopropyl β-d-1-thiogalactopyranoside typically for 3 h. Proteins were purified from the cell lysate by nickel-affinity chromatography (NAC, step 1). The bound fraction was subjected to isocratic fractionation on Superose-12 column (step 2, Cytiva Life Sciences) and HRV-3C protease cleavage (step 3, purchased from Sigma-Aldrich) or TEV protease (produced in-house^{48 (link)},) overnight at 4 °C followed by repeating NAC and step 2 in a final buffer of 25 mM Tris-HCl, pH 7 or 7.6, 150 mM NaCl and 1 mM TCEP (buffer A). The full-length wild-type (MPro^WT) was expressed and purified similar in strategy to that described previously except for substituting the fusion partner GST with maltose binding protein (MBP) followed by a 36 amino acid spacer sequence corresponding to the immunoglobulin binding domain B1 of protein G (ΔGB1). Peak fractions were pooled and concentrated to the desired concentration and stored in aliquots at −30 °C and for long term storage at −80 °C. Purity was verified both by SDS-PAGE on 4–20% gradient mini-protean TGX precast gel (Bio-Rad) and electrospray ionization mass spectrometry.

Molecular mass of MPro^1–199 and ⁺²⁵MPro^{1–199(C145A)} was estimated by analytical SEC with in-line MALS (DAWN Heleos-II, Wyatt Technology Inc., Santa Barbara, CA), refractive index (Optilab T-rEX, Wyatt Technology Inc.) and UV (Waters 2487, Waters Corporation, Milford, MA) detectors. Sample (125 µl) was applied onto a pre-equilibrated Superose-12 column (1.0 × 30 cm, Cytiva) and eluted at a flow rate of 0.5 ml/min in buffer A at 25 °C. Molecular mass was calculated using the Astra software provided with the instrument.