Following induction of the promoter by IPTG, no CpxP protein was detectable from cells transporting pTCP, by Coomassie Blue staining, or immunoblotting (Fig

Following induction of the promoter by IPTG, no CpxP protein was detectable from cells transporting pTCP, by Coomassie Blue staining, or immunoblotting (Fig. Our results support the hypothesis that periplasmic expression could be inhibited by sequence elements in the early coding transmission sequence region of expression could be inhibited by the formation of mRNA secondary structures. When other well-characterized transmission sequences were fused to the mature CpxP, efficient production of functionally active CpxP was found. Finally, this study enabled us to produce large amounts of CpxP in the bacterial periplasm. Results Cytoplasmic versus periplasmic expression The gene, either with or without its own transmission sequence, was cloned under the control of the promoter around the pTrc99A expression vector, and a strain was transformed with the producing pTCP and pTCPss plasmids. Following induction of the promoter by IPTG, no CpxP protein was detectable from cells transporting pTCP, by Coomassie Blue staining, or immunoblotting (Fig. 1). In contrast, the cytoplasmic production of CpxP in cells transporting pTCPss resulted in high protein levels. This observation led us to consider that CpxP could be rapidly degraded by proteases when produced with its transmission sequence and more likely by periplasmic DegP, as was previously shown in the presence of P pilus subunits (Isaac et al. 2005). However, the expression from pTCP in a strain did not improve the steady-state level RFC4 of CpxP in the periplasm (data not shown). Therefore, we hypothesized that this dramatic difference of the CpxP level observed BPN-15606 between cytoplasmic and periplasmic expression might be due to effects exerted at the translational level. Since both mRNAs transcribed from pTCP and pTCPss contained the same 5 untranslated region but differed in the translational BPN-15606 initiation region, we performed two units of experiments to investigate whether this crucial sequence downstream of the start codon prevents the efficient production of CpxP in the periplasm. Open in a separate window Physique 1. Cytoplasmic versus periplasmic production of CpxP. (under promoter control. (mRNA BPN-15606 sequence in the region downstream of the start codon, silent base substitutions were designed to minimize both GC content and base-pair probability by using the knowledge-based software ProteoExpert (Fig. 2A). By this methodology, recently developed for improving the translational efficiency of mRNAs in cell-free expression (Voges et al. 2004), high protein levels from an in the beginning poorly expressed gene have been achieved in cells (Betton 2004). The first set M1 of silent base mutations was launched into the pTC*P plasmid, creating a CpxP signal sequence derivative by maintaining the same amino acid sequence as that encoded by pTCP. Following induction, spheroplasts of cells transporting pTCP or pTC*P were fractionated and periplasmic fractions were analyzed by SDS-PAGE (Fig. 2B). Amazingly, the expression of with the mutated transmission sequence resulted in the presence of large amounts of CpxP in the periplasm. Even though quantification of this effect could not be realized because of the absence of CpxP in the wild-type condition, the four base substitutions dramatically increased the production of the protein. However, the precise contribution of specific sequence motifs or features in the mRNA cannot be estimated by the software. Nevertheless, we investigated whether base-pair probability in the mutated transmission sequence could help to explain this positive effect on expression. A putative mRNA secondary structure was predicted for the gene encoding the transmission sequence BPN-15606 by the Mfold program (Fig. 3A). Among the four base substitutions that increased the production of CpxP, three may impact base pairing of this hypothetical stemCloop structure. To test whether this putative mRNA secondary structure would be disrupted by an increase of heat, and thus enhancing the translation efficiency, we compared the level of CpxP in cells produced at 37C and at 43C (Fig. 3B). While the steady-state level of CpxP produced with the wild-type transmission sequence remained undetectable even at high temperature, a twofold increase between 37C and 43C was found for the CpxP with the optimized transmission sequence. This result is usually consistent with a heat destabilization of a mRNA structure that limits the synthesis of CpxP. Open in a separate window Physique 2. Silent mutations enhancing the periplasmic production of CpxP. (proposed by ProteoExpert are ranked as the indicated order with M1 having the highest predicted expression score. (periplasmic proteins, and it was recently shown that their transmission sequences can promote post- and cotranslational precursor translocation,.