FIG. 5.

Complexes of EcfA1A2T with FolT, PanT, and RibU. E. coli BL21 containing pLacI-RARE2 was used as the host. The EcfA1A2T module with wild-type (WT) and mutant (R184E, R225E, R184/225E) ecfT genes was produced from a plasmid (resulting in deca-His-tagged EcfA1, untagged EcfA2, and FLAG-tagged EcfT) in the presence of another plasmid encoding FLAG-tagged FolT, PanT, or RibU. Membranes of the recombinants were isolated and solubilized with dodecyl-β-d-maltoside. Solubilized material was chromatographed on immobilized Ni-NTA. Enriched proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes. The membranes were probed with monoclonal anti-oligo-His or anti-FLAG antibody-alkaline phosphatase conjugates. The marker proteins (left lane in each panel) had molecular masses of (from top to bottom) 37, 25, and 20 kDa.