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 chloramphemcol
acetyltransferase (CAT) as a model system, several factors determining 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 product10n
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 pr10r
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
