Induction of recombinant gene expression in Escherichia coli using an alkaline pH shift.
A commonly used approach to control recombinant protein production in Escherichia coli utilizes the lambda pL promoter-operator and the lambda repressor. Inactivation of the lambda repressor function allows transcription to proceed. However, induction of the RecA-mediated cleavage of lambda repressor by the addition of nalidixic acid or inactivation of a temperature-sensitive lambda repressor by growth at the nonpermissive temperature can have detrimental effects on protein production. This paper describes the use of an alkaline shift in the pH of the growth medium that allows expression of genes from the pL promoter in a RecA-independent manner. This procedure results in high-level production of recombinant protein. The pH shift is performed in the stationary phase of cell growth, using culture volumes ranging from 1-1000 ml. This method can result in the production of over 15-fold more active protein than when using a temperature shift.
Poindexter
K
K
Department of Molecular Biology, Immunex Corporation, Seattle, WA 98101.
Gayle
R B
RB
D004274:DNA, Recombinant / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D011401:Promoter Regions, Genetic; D011938:Rec A Recombinases / Q000378:metabolism; D013696:Temperature
D004274:DNA, Recombinant; D011938:Rec A Recombinases