Two-cistronic expression plasmids for high-level gene expression in Escherichia coli preventing translational initiation inhibition caused by the intramolecular local secondary structure of mRNA.

Two-cistronic expression plasmids for the wild-type solubilized domain of porcine NADPH-cytochrome P450 reductase (PsCPR) gene in Escherichia coli were systematically constructed using a solubilized domain of porcine cytochrome b5 gene (Psb5 gene) or a derivative of it as the first cistron to examine their utility for second gene expression preventing the translational inhibition caused by the intramolecular local secondary structure of mRNA at the ribosome-binding site (RBS). The mRNAs from the plasmids lacking an RBS for the second cistron (SD2) accumulated very low levels of PsCPR, while those from the plasmids having an SD2 accumulated higher levels of PsCPR. The level of accumulation of PsCPR by the mRNA from plasmid pCbSD-T-CPR-3, which has an SD2 upstream of the termination codon of the first cistron, was higher than for those with an SD2 in the intercistronic region. The predicted intramolecular local secondary structures at the SD2 of mRNAs from these plasmids were stable enough to cause translational initiation inhibition. These results indicate that the use of a two-cistronic expression plasmid is an effective way to overcome translational initiation inhibition. Improved plasmids, pCP1 and pCP2P, were constructed from pCbSD-T-CPR-3. Using these plasmids, the solubilized donain of porcine NADH-cytochrome b5 reductase was also highly accumulated on prevention of the translational initiation inhibition. These plasmids are expected to be useful tools for the comprehensive high-level expression of heterologous genes in E. coli cells.