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 applicability in recombinant protein production was evaluated using a plasmid construct, pSM552-545C(-), in which the promoter and activator coding sequences of the cad operon were inserted into the upstream region 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 range can be as high as 200-fold. The effects of several cultivation parameters, including pH, temperature, induction cell density, and inoculum size, were systematically examined. The practical application of this expression system to high level production of recombinant proteins was successfully demonstrated using a rich medium, superbroth. An extremely high recombinant protein productivity at a value of approximately 1.4 g/L with a specific expression level as high as 35% of total cellular protein can be obtained in a simple batch cultivation. The behavior 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 activity at a dilution rate of approximately 0.4 h(-1)was observed. (c) 1995 John Wiley & Sons, Inc.
Chou
C H
CH
Department of Chemical Engineering, Institute of Biosciences and Bioengineering, Rice University, P.O. Box 1892, Houston, Texas 77251-1892.
Aristidou
A A
AA
Meng
S Y
SY
Bennett
G N
GN
San
K Y
KY