Effect of chemically-induced, cloned-gene expression on protein synthesis in E. Coli.
Earlier experiments in our lab investigated the metabolic limitations of cloned-gene expression in bacterial cells (for over-production of beta-lactamase). These experiments showed that the steady-state concentration of ribosomal RNA decreased upon plasmid amplification while both the synthesis rate and steady-state beta-lactamase mRNA level increased significantly. This appeared to indicate substantial limitation exist within the transnational machinery of the bacterial cell at high copy numbers. To establish the generality of this phenomenon, the impact increasing protein expression from pa 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, beta-galactosidase expression in chemostat cultures operated at specific growth rates of 0.6 h(-1) was induced with IPTG such that enzyme activity was varied over a 460-fold range. When fully induced beta-galactosidase protein production represented 14 wt % of total cell protein. As transcription was induced, the synthesis rate of the beta-galactosidase mRNA increased 42-fold while the steady-state level of beta-galactosidase mRNA increased only fourfold. This indicates stability may play a larger role for beta-galactosidase expression with a strong promoter than seen with beta-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 eRNA, transnational limitations are again suggested for the chemically induced transcription system.
Wood
T K
TK
Department of Chemical Engineering, North Carolina State University, Raleigh, North Carolina 27695-7905, USA.
Peretti
S W
SW