Analysis and optimization of recombinant protein production in Escherichia coli using the inducible pho A promoter of the E. coli alkaline phosphatase

Abstract

The use of the promoter of the Escherichia coli alkaline phosphatase gene (pho A promoter) allows an efficient and strictly controllable expression of recombinant proteins in E. coli. As an example, the expression 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 plasminogen 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 analyzed 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 production cultures with higher (up to 10%) amounts of preculture the plasmid loss could be reduced and the yield of heterologous protein increased.