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 (RES) indicates that the control of expression by base composition is mediated by the secondary structure of the RES. An unfolded state of the RES 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 RES 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. (C) 1995 Academic Press, Inc.