FIG. 2.
Negative effect of the bmlP1 3′ flanking region on BmlP1 production in E. coli. (A) The expression vector pKK223-3-derived plasmids, pGS99 and pGS98, contain the bmlP1 coding region alone and the bmlP1 coding region plus the 500-bp 3′ flanking region, respectively. In the pKK223-3-derived plasmid pGS520, the 490-bp bmlP1 3′ flanking region is fused in the reverse orientation to the 3′ end of bmlP1. A 360-bp internal region (from positions +174 to +532 relative to the translational start site) of the B. megaterium mbgA gene is fused in both orientations to the 3′ end of bmlP1 in plasmid pGS99 to generate plasmids pGS496 and pGS502. Numbers of base positions are given relative to the translational start site of bmlP1. S/D, putative ribosome-binding site of bmlP1; Ptac, the tac promoter. (B) E. coli cells carrying the above plasmids were grown at 37°C to an absorbance at 600 nm of 0.5 and then treated with 0.3 mM IPTG for 2 h or left untreated. Whole-cell protein extracts from equal numbers of cells were subjected to SDS-PAGE. (C) Whole-cell protein extracts used for panel B were subjected to SDS-PAGE followed by immunoblotting with polyclonal anti-BmlP1 antibodies as the probe.