CHARACTERIZATION OF A PH-INDUCIBLE PROMOTER SYSTEM FOR HIGH-LEVEL EXPRESSION OF RECOMBINANT PROTEINS IN ESCHERICHIA-COLI

A pH-inducible promoter system was characterized and its potential app licability in recombinant protein production was evaluated using a pla smid construct, pSM552-545C(-), in which the promoter and activator co ding sequences of the cad operon were inserted into the upstream regio n of a lacZ' reporter gene. Graded gene expression levels with respect to culture pH between 8.0 and 5.5 were observed and the induction ran ge can be as high as 200-fold. The effects of several cultivation para meters, including pH, temperature, induction cell density, and inoculu m size, were systematically examined. The practical application of thi s expression system to high-level production of recombinant proteins w as successfully demonstrated using a rich medium, superbroth. An extre mely high recombinant protein productivity at a value of approximately 1.4 g/L with a specific expression level as high as 35% of total cell ular protein can be obtained in a simple batch cultivation. The behavi or of this expression system was further investigated using chemostat cultures. An uncommon relationship between the volumetric or specific recombinant protein activity and the dilution rate, with a maximal act ivity at a dilution rate of approximately 0.4 h(-1), was observed. (C) 1995 John Wiley and Sons, Inc.