Fig. 3.
Utilization of the MATE system for the transport of a small peptide with an N-terminal 6xHis to the outer membrane. E. coli BL21 cells were grown in LB medium to A600 nm=0.5. Protein expression was induced by the addition of 1 mM IPTG, and the cells were harvested after 1.5 h. Outer membrane proteins were isolated according to the modified method of Park et al. (32): a) SDS-PAGE of outer membrane proteins including the reference outer membrane proteins OmpF and OmpA; b) Western blot of outer membrane proteins with an antibody against 6xHis. M=PageRuler prestained protein marker, 1=cells with negative control plasmid, expressing a 74.5-kDa sized fusion protein containing the AIDA-I autotransporter with a single- -chain antibody fragment as passenger [pST005, (38)], 2=cells with empty vector (pJExpress-401), 3=cells with pMATE-MT004 without the addition of IPTG, 4=cells with pMATE-MT004 with 1 mM IPTG for the induction of protein expression. The arrow indicates the band associated with the MATE fusion protein