Nucleic Acids Research, 1985, Vol. 13, No. 9 3101-3110
© 1985
Articles |
Up-promoter mutations in the lpp gene of Escherichia coli
Department of Biochemistry, State University of New York Stony Brook, NY 11794, USA
Received March 4, 1985. Accepted April 5, 1985.
The promoter of the gene for the major outer membrane lipoprotein, the most abundant protein in Escherichia coli, is considered to be one of the strongest promoters in E. coli. The nucleotide sequences of the –10 and the –35 regions of the 1pp promoter were altered in a step-wise manner to conform to their respective consensus sequences by synthetic oligonucleotide-directed site-specific mutagenesis. The mutated promoters were then fused to the lacZ gene to measure promoter activity. The ß-galactosidase activity increased approximately 1.9 and 2.4 fold when the –10 region (AATACT) was altered to TATACT(P1) 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(R1) and TTGACA (consequence 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 lpp promoter in an expression cloning vehicle pIN-III-ompA, staphylococcal nuclease A was produced at a level of approximately 47% of the total cellular protein.
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