Low concentration of inducer favors production of active form of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Escherichia coli.

Expression of chicken and rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, in Escherichia coli encountered two common problems: the chicken enzyme was liable to proteolysis and the rat enzyme was prone to form inclusion bodies. Reducing the rate of protein synthesis by lowering either growth temperature or isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration alleviated these two problems. Growth at 22 degrees C was optimum for expression of both enzymes. The optimum range of IPTG concentration for expression was 0.1-1 microM for the chicken liver bifunctional enzyme and 10 microM for rat liver enzyme. The components of growth medium also influenced the production. Compared with Luria-Bertani medium, an enriched medium-tryptone-phosphate medium-tripled the production of the active enzymes. Addition of glucose (0.2%) doubled the expression level of active chicken liver enzyme, but reduced the production of active rat liver enzyme to half the maximal level, while the phosphate in tryptone-phosphate medium had no effect on the production of the two enzymes.