Studies, optimization, and application of protein excretion in Escherichia coli.

摘要

Induced expression of a 49 kD Cellulomonas fimi exoglucanase (Exg) encoded by the cex gene using the Tac-cassette system was lethal to its E. coli host. Replacement of the cassette tac promoter by the weaker lacUV5 promoter resulted not only in higher levels of cell viability and plasmid stability, but also in better yields of extracellular Exg, as measured by both total activity and specific activity. Efforts were then made to explore the cause of the lethal effect of hyper-expression of extracellular Exg on E. coli. Deletants of Exg lacking either the cellulose-binding site or the active site were as toxic as the full-length Exg, indicating that neither the binding property nor the enzymatic activity of Exg was responsible for the lethality. Results from Northern-blot analysis revealed that enhanced production of the cex transcript under the control of an induced tac promoter did not cause cell death. Western-blot analysis of cell lysate samples indicated that the induced expression, employing the Tac-cassette, of the cex gene fused to the ompA leader sequence resulted in a cell-bound accumulation of Exg as an OmpA-signal peptide/Exg hybrid precursor. Further studies using cell-fractionation techniques revealed that the precursor was associated with the inner-membrane fraction. This association was shown to be affected by the ultimate destination, the expression level and the size of Exg. A model, designated "translocation saturation" was proposed to account for the lethal effect of Exg expression on E. coli cells. It is suggested that saturation of the translocation machinery in the inner membrane by the OmpA-signal peptide/Exg precursor leads to a blockage of transport of other proteins, including some essential for cell viability, across the inner membrane, thereby resulting in cell death. The model was supported by the observation that over-expression of Exg as an intracellular product was not lethal to the host. The ability of the LacUV5-cassette to promote extracellular expression of heterologous proteins in E. coli was further shown by its application to the production of human epidermal growth factor on a large scale.