THE EFFECTS OF PLASMID CONTENT, TRANSCRIPTION EFFICIENCY, AND TRANSLATION EFFICIENCY ON THE PRODUCTIVITY OF A CLONED GENE PROTEIN IN ESCHERICHIA-COLI

In order to investigate how plasmid content, transcription efficiency, and translation efficiency affect the productivity of a cloned gene protein, a new vector (pPLc-RP4.5) was constructed. The vector has PL promoter, lacZ as a structural gene and 4.5S RNA gene between PL promoter and lacZ gene. We took advantage of the characteristic that the 4.5S RNA is accumulated inside E. coli cells and can be quantitatively measured. A two-stage continuous culture system in combination with a temperature-sensitive gene switching system was used to study the performance of the recombinant fermentation. It was found that the plasmid content as varied by the dilution rate in the production stage showed a different pattern from that in the growth stage. The results showed that promoter strength had a greater influence on the overall gene expression efficiency of a cloned gene than the plasmid content, and the overall gene expression efficiency was largely dependent upon translation efficiency when a multicopy plasmid (pBR322 derivative and rop-) and a strong promoter (PL) were used to express a heterologous protein in E. coli.