Guanidinium hcl

Medium and buffer components were purchased from Fisher Chemical. Amino acids, casAmino acids (acid-hydrolyzed), copper sulfate, ferrous ammonium sulfate, cytochrome c, horseradish peroxidase (HRP), Amplex Red, hydrogen peroxide solution (30% w/w), bovine catalase, ascorbic acid, ovalbumin, β-mercaptoethanol, N-acetylcysteine, bovine superoxide dismutase, reduced (GSH) and oxidized (GSSG) glutathione, E. coli alkaline phosphatase (catalog P5931), ascorbate, p-nitrophenylphosphate, zinc(II) diacetate, guanidinium HCl, citraconate, diethylenetriaminepentaacetic acid (DTPA), EDTA, NADH, NAD⁺, recombinant E. coli formate dehydrogenase (FDH), rabbit L-lactate dehydrogenase, xanthine, bovine xanthine oxidase, and antibiotics were from Sigma Aldrich. Coomassie reagent was from Thermo Scientific. Formyl-Met-Ala-Ser was from Bachem.

All recombinant proteins were expressed in E. coli expression strain BL-21 ΔTreA and purified on Ni-NTA resin (Thermo Fisher Scientific, Ottawa, Canada), as described [25 (link)]. In short, the protein expression was induced with 0.5 mM of IPTG (isopropyl-β-D-1-thiogalactopyranoside) (UBP Bio, Aurora, CO) for 3 h at 37 °C. Bacterial pellets expressing recombinant proteins were re-suspended in 6 M guanidinium buffer, lysed by sonication and loaded on equilibrated Ni-NTA resin. Proteins were refolded on resin during washing steps containing gradually decreasing guanidinium-HCl (Sigma-Aldrich, Oakville, Canada) concentrations and eluted in an elution buffer containing 500 mM of imidazole (Sigma-Aldrich, Oakville, Canada). Finally, samples were dialyzed against 1 L of sodium maleate buffer (Sigma-Aldrich, Oakville, Canada) (50 mM, pH 6) with Snakeskin (Fisher Thermo Fisher Scientific, Ottawa, Canada) for 24 h at 25 °C and protein concentration determined by Qubit assay (Thermo Fisher Scientific, Ottawa, Canada).