ANALYSIS AND OPTIMIZATION OF RECOMBINANT PROTEIN-PRODUCTION IN ESCHERICHIA-COLI USING THE INDUCIBLE PHO-A PROMOTER OF THE ESCHERICHIA-COLI ALKALINE-PHOSPHATASE

The use of the promoter of the Escherichia coli alkaline phosphatase g ene (pho A promoter) allows an efficient and strictly controllable exp ression of recombinant proteins in E. coli. As an example, the express ion of a gene coding for a fusion protein consisting of the N-terminal part of the E. coli alkaline phosphatase linked via four collagenase recognition;sequences to the finger domain of the human tissue plasmin ogen activator was investigated. Expression was measured at the level of protein production. The time course of phosphate consumption, cell growth, plasmid stability, and recombinant protein synthesis was analy zed for this system. It was found that the expression regulated by the pho A promoter was induced at a phosphate concentration of below 0.05 mM. In the induced state of the pho A promoter the cells had a strong tendency to delete the recombinant plasmid. By inoculating the produc tion cultures with higher (up to 10%) amounts of preculture the plasmi d loss could be reduced and the yield of heterologous protein increase d.