Recombinant Human Proinsulin Expression in E. Coli by Altering 5ʹ Untranslated and Translated Region

Messenger RNA initiate the process of translation, by transferring the code of DNA to transfer RNA. A poly purine rich sequence called Shine-Dalgarno sequence help to determine the position of the start codon, the Shine-Dalgarno sequence is different because it allows the ribosome to be built at an interior position on the mRNA through direct binding to this sequence. Ribosomal binding site at the 5’end of translation initiation site used to bind mRNA secondary structure, distance between RBS and start codon effects translational efficiency of a gene. In this study we change the distance between ribosome binding site and start codon to get the different ratios of translational expression. For this purpose, proinsulin gene cloned in pET21a vector, the distance between the binding site and starting codon has been retained to 8 nucleotides. At this distance between ribosome binding site (RBS) and start codon, the expression of proinsulin was 30% of total cellular proteins. When there are 10 nucleotides between, expression decreased up to 2-4%. With 12 nucleotides between RBS and start codon, expression further decreased up to 1-2%. As these attempts are made random, so by checking the mRNA secondary structures by M-fold showed a binding between ribosome binding site and start of proinsulin gene. Another attempt was made by incorporating ten different nucleotides at the start of proinsulin gene to make the secondary structure of mRNA less stable (ΔG=-5.5) does not significantly alter the expression of proinsulin in BL21 codon plus cells. There are other controlling factors of protein expression i.e., metabolic instability, rapid degradation of mRNA or accumulation of protein may downregulate expression of mRNA.