Enhancing the protein production levels in Escherichia coli with a strong promoter.

ABSTRACT

In biotechnology, the use of Escherichia coli for recombinant protein production has a long tradition, although the optimal production conditions for certain proteins are still not evident. The most favorable conditions for protein production vary with the gene product. Temperature and induction conditions represent parameters that affect total protein production, as well as the amount of soluble protein. Furthermore, the choice of promoter and bacterial strain will have large effects on the production of the target protein. In the present study, the effects of three different promoters (T7, trc and lacUV5) on E. coli production of target proteins with different characteristics are presented. The total amount of target protein as well as the amount of soluble protein were analyzed, demonstrating the benefits of using a strong promoter such as T7. To understand the underlying causes, transcription levels have been correlated with the total amount of target protein and protein solubility in vitro has been correlated with the amount of soluble protein that is produced. In addition, the effects of two different E. coli strains, BL21(DE3) and Rosetta(DE3), on the expression pattern were analyzed. It is concluded that the regulation of protein production is a combination of the transcription and translation efficiencies. Other important parameters include the nucleotide-sequence itself and the solubility of the target protein.