Expression of synthetic calcitonin genes in plasmid vectors containing tandemly repeated non-overlapping ribosome binding sites

Abstract

1. 1. Expression plasmids containing non-overlapping tandemly repeated ribosome binding sites (RBS) were constructed in order to stabilize mRNA and enhance translation. 2. 2. Two synthetic genes (human calcitonin tetramer gene and a fusion gene human γ-interferon-human calcitonin) were cloned in these vectors and the effect of multiplicity of Shine-Dalgarno (S/D) sequence on heterologous gene expression was studied. 3. 3. It was found that duplication and triplication of RBS had no effect on the stability of mRNA but led to a strong decrease in the level of recombinant protein and mRNA in the cell. 4. 4. Plasmids bearing four times repeated S/D sequences gave longer-lived mRNAs and maintained a level of protein and mRNA very close to the values obtained with a single S/D containing plasmids.