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. 1998 Feb;180(3):680–689. doi: 10.1128/jb.180.3.680-689.1998

FIG. 7.

FIG. 7

SDS-PAGE analysis of TMBP after expression in E. coli. (A) TMBP with the N-terminal cleavable signal sequence of E. coli (construct A in Fig. 1). Lanes: 1, uninduced E. coli cells harboring pRHo1000; 2, induced cells; 3, cytoplasmic extract of induced cells that were treated by cold osmotic shock; 4, same as lane 3 but the sample was heated to 80°C for 10 min; 5, periplasmic shock proteins of induced cells; 6, same as lane 5 but the sample was heated to 80°C for 10 min; 7, TMBP purified from the sample shown in lane 6; 8, TMBP isolated from T. litoralis membranes; St, molecular mass standards (from top to bottom: 94, 67, 43, and 30 kDa). (B) Cytoplasmic TMBP; its N terminus is the expected cleavage site after the E. coli signal sequence (construct B in Fig. 1). Lanes: 1, uninduced E. coli cells harboring pRHo1002; 2, induced E. coli cells; 3, cytoplasmic extract of induced cells; 4, same as lane 3 but the sample was heated to 80°C for 10 min; 5, purified periplasmic TMBP (construct A in Fig. 1); 6, TMBP isolated from T. litoralis; St, as for panel A. (C) Native precursor TMBP from T. litoralis. The protein was lipid modified and membrane bound (construct C in Fig. 1). Lanes: 1, membranes from E. coli harboring pRHo1001 solubilized in octyl-β-glucoside and induced for the expression of the native precursor TMBP; 2, same as lane 1 but the sample was heated for 10 min to 80°C; 3, purified TMBP from T. litoralis; 4, purified periplasmic TMBP endowed with the E. coli signal sequence; St, as for panel A. (D) Western blot of TMBP from T. litoralis membranes and from the different constructs expressed in E. coli. Lanes: 1, whole E. coli cells harboring pRHo1000 encoding the hybrid protein with the E. coli signal sequence; 2, whole cells harboring pRHo1002 encoding the hybrid protein without the E. coli signal sequence; 3, whole cells harboring pRHo1001 encoding the intact T. litoralis protein; 4, membrane preparations of T. litoralis uninduced for the transport system; 5, membrane preparations of T. litoralis induced for the transport system; St, protein standards; 6, same as lane 1 (whole cells harboring pRHo1000); 7, French pressure cell extract of cells harboring pRHo1000; 8, periplasmic proteins isolated from cells harboring pRHo1000.