High-level expression of prochymosin in Escherichia coli: effect of the secondary structure of the ribosome binding site.

Regulation of the expression of prochymosin cDNA in Escherichia coli at the translational level was studied by mutating the regions between the Shine-Dalgarno (SD) sequence and the initiation codon and upstream of the SD signal. Results revealed that expression plasmids with a distance of 7-10 bp from SD to ATG have the potential to be expressed at higher levels. However, an approximately 20-fold variation in expression was observed with plasmids harboring different base composition but identical distance in the spacer. Analysis of the predicted secondary structure of ribosome binding sites (RBS) indicates that the control of expression by base composition is mediated by the secondary structure of the RBS. An unfolded state of the RBS is required for high expression. Therefore, a vector for enhanced translation can be designed and constructed via prediction of the secondary structure of the proposed RBS and mutagenesis. Based on this strategy, high-level expression of prochymosin, up to 39% of the total cellular proteins, was achieved. The 9-base sequence proposed by Olins and Rangwala as a translational enhancer did not exhibit an additive effect on prochymosin expression. This is probably because the affinity of the SD sequence used in this study to 16S rRNA is strong enough that no additional element is required to facilitate the formation of the translation initiation complex.