FIG. 5.

(A) SDS-PAGE gel showing selectivity of amylose-agarose affinity chromatography for Sus OMPs. Molecular markers are shown to the left of the blot. Each protein labeled on the right was confirmed by immunoblots using antisera directed against the corresponding protein. Lanes: 1, membrane proteins of B. thetaiotaomicron 5482; 2, n-octyl-β-d-glucopyranoside-solubilized membrane proteins; 3, wash fraction of n-octyl-β-d-glucopyranoside-solubilized membrane proteins after loading onto an amylose-agarose column; 4, maltose eluant fraction of n-octyl-β-d-glucopyranoside-solubilized membrane proteins. (B) Immunoblots showing changes between various mutants in amylose binding activity of SusC, SusD, and SusE. The protein is labeled on the right of the blot, with the mutant strains expressing the corresponding protein labeled to the left of the blot. Lanes: 1, membrane fraction of the mutant; 2, n-octyl-β-d-glucopyranoside-solubilized membrane fraction of the mutant; 3, wash fraction of n-octyl-β-d-glucopyranoside-solubilized membrane proteins of the mutant after loading onto amylose-agarose column; 4, maltose eluant of n-octyl-β-d-glucopyranoside-solubilized membrane fraction of mutants. Although not evident in the figure, lane 4 of the immunoblot for mutant ΩsusE did show a low level of SusC. The designation ΩsusD + ΩsusC(pSDC27) indicates that solubilized membrane fractions from a strain producing only SusD and one producing only SusC were incubated together overnight before being loaded on the column.