INCREASED EFFICIENCY OF ALKALINE-PHOSPHATASE PRODUCTION LEVELS IN ESCHERICHIA-COLI USING A DEGENERATE PELB SIGNAL SEQUENCE

To obtain an expression vector that will optimize secretion of protein s with disulfide bridges in Escherichia coli, we fused the phoA gene, encoding the bacterial alkaline phosphatase (PhoA), to the sequence en coding the pectate lyase B signal sequence (PelBSS), We used an extens ively degenerate pelBSS with silent mutations to study their effects o n the production level and activity of PhoA, 11 representative clones differed by a factor of five between the lowest and the highest level of activity, and by a factor greater than seven for the production lev els. The efficiency of translocation seems to be the result of an equi librium between production and secretion levels that favours the secre tion of active PhoA according to the competence of the fusion protein being translocated. Free energy calculations and the predicted mRNA se condary structures of the translation initiation regions showed that t he high stability of the secondary structure decreased production and secretion levels of PhoA and vice versa. A stem-loop encompassing the degenerate positions downstream from the AUG start codon appears to be responsible for the differences in the production levels.