Nucleic Acids Research

The promoter of the gene for the major outer membrane llpoprotein, the most abundant protein in Escherichia coll. is considered to be one of the strongest promoters in 12. coll. The nucleotide sequences of the -10 and the -35 regions of the Ipp promoter were altered in a step-wise manner to conform to their respective consensus sequences by synthetic oligonucleotide-dlrected site-specific mutagenesis. The mutated promoters were then fused to the lacZ gene to measure promoter activity. The 8-galactosidase activity increased approximately 1.9 and 2.4 fold when the -10 region (AATACT) was altered to TATACT(Pl) and TATAAT (consensus sequence; P2) , respectively. Similarly, it Increased approximately 1.2 and 4.2 fold, when the -35 region (TTCTCA) was altered to TTCACA(Rl) and TTGACA(con8ensus sequence; R2), respectively. When the mutations at the -10 and -35 regions were combined, the overall improvement of the promoter activity for R2-P1 was 4.0 fold over that of the wild-type promoter, while it was only 2.5 fold for R2-P2. These results indicate that substantial improvement of the promoter activity can be achieved by changing either of the two key regions to their respective consensus sequences. However, the complete conformity to consensus sequences at both regions does not necessarily result In the highest activity. With use of the improved Ipp promoter in an expression cloning vehicle pIN-III-ompA, staphylococcal nuclease A was produced at a level of approximately 47Z of the total cellular protein.