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Hosseini oula S K, Saeedinia A R, Zeinoddini M. The Modified Recombinant Proinsulin: A Simple and Efficient Way to Produce Insulin Glargine in E. coli. Res Mol Med (RMM) 2021; 9 (3) :189-196
URL: http://rmm.mazums.ac.ir/article-1-439-en.html

Background: Recombinant insulin glargine, a long-acting analog of insulin, is expressed as proinsulin in the host cell and, after purification and refolding steps, is processed to mature insulin by using trypsin and carboxypeptidase B. Because of several internal residues of arginine and lysine in the proinsulin B
and C chains, several unwanted products are formed after treatment with these enzymes. To overcome this problem, we introduced three thrombin recognition sites into the proinsulin encoding sequence.

Materials and methods: After the design, the modified proinsulin encoding sequence containing the 5′ His-Tag tail and three thrombin recognition sites located between the His-Tag and B chain, B and C chains, and C and A chains, respectively, was synthesized by overlap extension Polymerase Chain Reaction (PCR) using seven specific primers in multiple sequential PCR reactions. The final amplified fragment was cloned in the pGEM-5zf vector by the EcoRV enzyme. After sequencing, the modified proinsulin encoding sequence was subcloned into the pET-26b(+) vector using NdeI and XhoI enzymes. Finally, the modified proinsulin was expressed in E. coli BL-21(DE3) by induction with Isopropyl β-d-1-thiogalactopyranoside (IPTG).

Results: The accuracy of the synthesized modified proinsulin sequence was confirmed by DNA sequencing. The modified proinsulin cloning was evaluated by PCR with specific primers and digestion with specific restriction enzymes. In this study, the modified proinsulin protein was expressed up to 40%. The modified proinsulin protein expression was assessed using sodium dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) and western blotting.

Conclusions: This modified proinsulin can be used to easily and efficiently produce insulin glargine without any impurities after processing with thrombin in one step in a nickel chromatographic column.