the modified recombinant proinsulin: a simple and efficient way to produce insulin glargine in e. coli

Background: recombinant insulin glargine, a long-acting analog of insulin, is expressed as proinsulinin the host cell and, after purification and refolding steps, is processed to mature insulin by using trypsinand carboxypeptidase b. because of several internal residues of arginine and lysine in the proinsulin band c chains, several unwanted products are formed after treatment with these enzymes. to overcomethis problem, we introduced three thrombin recognition sites into the proinsulin encoding sequence.materials and methods: after the design, the modified proinsulin encoding sequence containingthe 5′ his-tag tail and three thrombin recognition sites located between the his-tag and b chain, band c chains, and c and a chains, respectively, was synthesized by overlap extension polymerasechain reaction (pcr) using seven specific primers in multiple sequential pcr reactions. the finalamplified fragment was cloned in the pgem-5zf vector by the ecorv enzyme. after sequencing, themodified proinsulin encoding sequence was subcloned into the pet-26b(+) vector using ndei andxhoi enzymes. finally, the modified proinsulin was expressed in e. coli bl-21(de3) by inductionwith isopropyl β-d-1-thiogalactopyranoside (iptg).results: the accuracy of the synthesized modified proinsulin sequence was confirmed by dnasequencing. the modified proinsulin cloning was evaluated by pcr with specific primers anddigestion with specific restriction enzymes. in this study, the modified proinsulin protein wasexpressed up to 40%. the modified proinsulin protein expression was assessed using sodium dodecylsulfate–polyacrylamide gel electrophoresis (sds-page) and western blotting.conclusion: this modified proinsulin can be used to easily and efficiently produce insulin glarginewithout any impurities after processing with thrombin in one step in a nickel chromatographic column.