Fig. 3.
Isolation of a recombinant [FeFe]-hydrogenase. (A) E. coli strain PK4854 (ΔiscR) was transformed with plasmids: pUNI-Tte-HydhisC and pSUtat-Sh-GX-EF and cultured in LB supplemented with 0.4% (w/v) glucose, 2 mM cysteine, 2 mM ferric ammonium citrate. Cells were harvested and lysed by sonication. Crude cell extract was loaded onto a HisTrap™ HP column and eluted by an imidazole gradient and fractions (‘IMAC elution’) were collected and separated by SDS–PAGE (14% w/v acrylamide). Each subunit was identified using tryptic peptide mass fingerprinting. (B) Identification of HydCHis by Western immunoblotting. (C) SDS–PAGE analysis of the synthetic enzyme following SEC-MALLS. The peak fraction from SEC-MALLS analysis was collected, concentrated and separated by SDS-PAGE (12% w/v polyacrylamide), followed by staining with Instant Blue™.