FIGURE 2.

Coexpression of Pro-σ^K(1–126)-His₆ and SpoIVFB in E. coli and effect on solubilization of SpoIVFB from membranes. A, E. coli cell and an expanded view of the inner (IM) and outer membrane (OM). Pro-σ^K(1–126)-His₆ associates peripherally with the inner membrane (45). The topology of SpoIVFB was determined previously (77). SpoIVFB has a C-terminal CBS domain, and in some experiments an extra TMS (cytTM) was added to its N-terminal end. B, solubilization of coexpressed proteins. E. coli bearing pYZ42 was grown in a fermentor and induced with IPTG to coexpress Pro-σ^K(1–126)-His₆ and SpoIVFB-TEV-FLAG₂ E44Q. The membrane (M) fraction from 5 g of cells was untreated or treated with the indicated detergent to solubilize proteins. After high-speed centrifugation, the supernatant was subjected to immunoblot analysis with anti-FLAG (top) or anti-His (bottom) antibodies. PC, n-dodecylphosphocholine; LMPC, 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine; DDAO, n-dodecyl-N,N-dimethylamine-N-oxide; LAPAO, 3-dodecylamido-N,N′-dimethylpropyl amine oxide. C, solubilization of enzyme alone. This is the same as in A, except pYZ50 was used to express cytTM-SpoIVFB-TEV-FLAG₂-His₆ alone, and immunoblot analysis was done with only anti-FLAG antibodies. A dashed white line was added to help visualize the demarcation between lanes 1 and 2. In both panels, representative results from at least two biological replicates are shown.