Figure 1. Pellets of E. coli strain BL21(DE3) expressing different FbpA proteins.

E. coli BL21(DE3) were freshly transformed with a pT7-7 plasmid carrying WT or mutant H. influenzae fbpA genes for expression of the indicated FbpA protein (WT, Q58L, H9Q, H9A, E57A, Y196A or Y195A). The cultures were corrected to A₆₀₀=1.0 and the cells from the cultures were collected by centrifugation prior to photographing (A, B, D and E). (A and D) Bacterial pellets from cultures with exogenous iron added for 2 h before centrifugation. (B and E) Bacterial pellets from cultures without exogenous iron added. Periplasmic proteins were isolated from pellets in (B) and (E) by the modified osmotic procedure described in the Experimental section and a 10 μl sample of the periplasmic fraction was subjected to SDS/PAGE and stained with Coomassie Blue. The results are shown in (C) and (F).