Anaerobic Purification of Hydrogenases

E. coli strains BL21(DE3) and BL21(DE3) ΔiscR contained the pACYCDuet-1–hydGX–hydEF plasmid and one of the two pET-21(b) hydrogenase plasmids. Cells were grown in LB Miller growth medium supplemented with kanamycin (40 mg·L⁻¹; only when using the ΔiscR strain), chloramphenicol (25 mg·L⁻¹), ampicillin (100 mg·L⁻¹), 0.5% w/v glucose (∼25 mM), and 100 mM MOPS/NaOH (final pH of medium was 7.4). The ΔiscR strain contains a chromosomal substitution of the iscR gene with another gene conferring resistance to kanamycin [22] (link). 10–50 mL cultures were grown for investigating the effects of cell strains and substrates, while 50–250 mL cultures were grown for hydrogenase purification work. Initially, all cultures were grown aerobically at 25°C until an OD₆₀₀ of 0.3–0.5. They were then moved into an anaerobic glove box (Coy Laboratory Products) containing 98% N₂ and 2% H₂ prior to IPTG-based T7 RNA polymerase induction and heterologous protein expression. While ferric ammonium citrate (2 mM) was added to the growth medium prior to inoculation, both cysteine (2 mM) and sodium fumarate (25 mM) were added with IPTG (0.5 mM) within the anaerobic glove box. Cultures were sealed and incubated at 25°C for 16–24 hours following induction.
For investigating media formulations and protein expression by different strains, cells from 1 mL of culture were pelleted at 4,000×g and resuspended in 100 µL of anaerobic BugBuster® Master Mix lysis solution (Novagen) containing an additional 25 mM Tris/HCl (pH 8.0), 25 mM KCl, 3 mM sodium dithionite (DTH), 1 mM dithiothreitol (DTT), 2% v/v glycerol, 0.1% v/v Tween 20, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 2 µM resazurin as an oxygen indicator. After cell lysis (incubation at 25°C for 20 min), lysates were clarified by centrifugation at 14,000×g. Hydrogenase activities in cell lysates were measured using the methyl viologen reduction assay described below. Total protein content of lysates was determined using a commercial assay (Bio-Rad) based on the method of Bradford [42] (link), and the extent of heterologous protein expression was visualized using polyacrylamide gel electrophoresis with SDS-PAGE gels (Invitrogen).
Hydrogenase purifications were carried out while maintaining strict anaerobic conditions. After centrifugation and lysis as described above, approximately 1 mL of Strep-Tactin® Superflow® high capacity resin (IBA GmbH) was used per 50 mL of cell culture for purification. Wash and elution buffers contained the above lysis buffer additives excluding the BugBuster Master Mix and PMSF, and active hydrogenase was eluted with 2.5 mM D-desthiobiotin. Elution fractions were evaluated for active hydrogenase using the methyl viologen reduction assay, and fractions with high activity were pooled. Protein concentrations were measured with the Bradford assay, and hydrogenase iron content was measured using a ferrozine-based colorimetric assay [43] (link). Hydrogenase samples for IR spectroscopic studies were anaerobically concentrated to ∼100 µM using a 10 mL stirred cell and a 5 kD MWCO membrane (Amicon). Hydrogenase samples were not frozen prior to characterization and spectroscopic analysis.