Enhanced expression of cytochrome P450s from lac-based plasmids using lactose as the inducer.
The cytochrome P450 expression systems used in Escherichia coli are highly regulated and involve the use of the lac repressor to control expression. Induction in these systems utilizes the nonmetabolizable analog of lactose, isopropyl-beta-D-thiogalactopyranoside (IPTG), which is the most expensive compound required for an E. coli expression system. To determine if the natural inducer lactose could be used to induce cytochrome P450 expression we examined the expression of three P450 enzymes in E. coli using two different expression systems, pTrc99A and the T7-based PET22b vector. For both systems lactose was found to induce expression of active P450 to concentrations that exceeded the levels achieved with IPTG. A 20-liter fermentation of a P450 expression system in the pTrc plasmid in which lactose was used as the inducer resulted in 2.4 micromol P450/liter, with a total yield of 2 g of cytochrome P450. The use of lactose for protein expression in E. coli should be broadly useful for the inexpensive, large-scale production of heterologous proteins in E. coli.
Woyski
D
D
Department of Chemistry and Biochemistry, California State University, Fullerton 92834, USA.
Cupp-Vickery
J R
JR
D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007785:Lactose / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics
D007544:Isopropyl Thiogalactoside; D003577:Cytochrome P-450 Enzyme System; D007785:Lactose