Fig. 2.
Affinity tagging of formate hydrogenlyase components. (A) The affinity tags do not interfere with FHL activity. The parental strain (MG1655) and derivatives expressing hycBHis (Bhis), hycEHis (Ehis), hycFHis (Fhis), and hycGHis (Ghis) were grown anaerobically in LB media (pH 6.4) supplemented with 0.4% (wt/vol) glucose. Cells were harvested by centrifugation, washed twice in 50 mM Tris⋅HCl (pH 7.4), and intact cells then assayed for formate-dependent H2 evolution in a H2-sensing electrode as described (5, 47). Error bars represent the SEM, n = 3. (B) FHL Fe−S subunits remain membrane bound following cell lysis. Strains were grown under fermentative conditions before being harvested, washed, and fractionated in the absence of detergent. Equal proportions of crude extracts (CE), total membranes (TM), and soluble proteins (S) were separated by SDS-PAGE and challenged with an anti-His monoclonal antibody (Upper). Fractionation and loading control immunoblots toward the HybO integral membrane protein (Middle), and the soluble maltose binding protein (MalE, Lower) are included. (C) FHL can be isolated in a single step. Strains were lysed in a detergent mixture before extracts were applied to IMAC columns and bound proteins eluted and pooled. Approximately 10 μg of concentrated samples were separated by SDS-PAGE and stained with Instant Blue. All protein bands analyzed by tryptic peptide mass spectrometry are labeled: 1, positively identified as FdhF; 2, HycE; 3, HycG; 4, HycB; 5, HycF; and 6, predominantly fragments of HycE. (D) Isolated HycEHis can be identified by Western immunoblotting. A CE of MG059e1 (hycEHis) was prepared from a detergent mixture (6 g of cells in 40 mL solution) and applied to a 5-mL IMAC column. The unbound protein (column flow-through, FT) was collected in 40 mL total. Bound proteins were eluted in a 30-mL gradient of 50–1,000 mM imidazole, and 10 × 1 mL peak fractions were collected, seven of which were applied to 12% (wt/vol) SDS-PAGE. Separated proteins were transferred to nitrocellulose and challenged with an anti-His monoclonal antibody.