Process characterization of a novel cross-regulation system for cloned protein production in Escherichia coli.
A novel cross-regulation expression system has been shown previously to be very effective for regulated recombinant protein production. Earlier studies established that this system offers better control of basal expression and higher maximal induced expression than more traditional vectors. Using production of cloned chloramphenicol acetyltransferase (CAT) as a model system, several factors determining the performance of this system were examined. Specifically, the effects of varying induction times and inducer (IPTG) concentrations on cell growth and the rate of CAT production were examined. The CAT expression was maximally induced with at least 0.5 mM IPTG added at the midexponential growth phase. Specific CAT content (on a total protein basis) was correlated with the CAT mRNA level. CAT message levels were minimal preinduction and far above background postinduction, consistent with prior simulation results. Cessation of CAT accumulation as the culture entered the stationary phase coincided with a corresponding 10-fold decrease in the level of CAT mRNA which was likely caused by an increased mRNA degradation rate. Maintenance of significant CAT message levels with a concomitant 2-fold increase in CAT accumulation was achieved by extending cell growth in a fed-batch process.
Chen
W
W
Department of Chemical Engineering, California Institute of Technology, Pasadena 91125, USA.
Kallio
P T
PT
Bailey
J E
JE
D001426:Bacterial Proteins / Q000096:biosynthesis*; D015152:Blotting, Northern; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D003470:Culture Media; D004269:DNA, Bacterial / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D009693:Nucleic Acid Hybridization; D012329:RNA, Bacterial / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis
D001426:Bacterial Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D015500:Chloramphenicol O-Acetyltransferase