Effect of chemically-induced, clonedgene expression on protein synthesis in

Earlier experiments in our lab investigated the metabolic limitations of cloned-gene expression in bacterial cells (for over-production of p-lactamase). These experiments showed that the steady-state concentration of ribosomal RNA decreased upon plasmid amplification while both the synthesis rate and steady-state p-lactamase mRNA level in-creased significantly. This appeared to indicate substantial limitations exist within the translational machinery of the bacterial cell at high copy numbers. To establish the gener-ality of this phenomenon, the impact of increasing protein expression from a plasmid by chemically inducing a strong promoter while maintaining constant copy number has been investigated. A plasmid has been constructed which contains the lacZ gene under control of the tac promoter and contains the parB stability locus to maintain plasmid stability. Using this vector, p-galactosidase expression in chemostat cultures operated at specific growth rates of 0.6 h- ’ was induced with IPTG such that enzyme activ-ity was varied over a 460-fold range. When fully induced, p-galactosidase protein production represented 14 wt % of total cell protein. A s transcription was induced, the synthe-sis rate of the p-galactosidase mRNA increased 42-fold while the steady-state level of p-galactosidase mRNA in-creased only fourfold. This indicates mRNA stability may play a larger role for p-galactosidase expression with a strong promoter than seen with p-lactamase production in the elevated copy number system. Furthermore, rRNA synthesis rates increased at high expression rates as seen in the copy number experiments. However, unlike the amplified-plasmid system, the steady-state levels of rRNA increased as well. Since the total protein levels closely followed the steady-state level of rRNA, translational limita-tions are again suggested for the chemically induced-transcription system. Key words: Escherichia coli protein synthesis metabolic limitations cloned-gene expression p-galactosidas