The modified recombinant proinsulin: a simple and efficient route to produce insulin glargine in E. coli

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Article Type:
Research/Original Article (دارای رتبه معتبر)
Abstract:
Background

Recombinant insulin glargine, a long-acting analogue of insulin, is expressed as proinsulin in host cell and after purification and refolding steps cleaved to active insulin by enzymatic digestion using trypsin and carboxypeptidase B. Since the proinsulin's B and C chains have several internal arginine and lysine residues, a number of impurities are generated following treatment with these enzymes. To overcome this problem, we introduced three thrombin recognition sites into proinsulin encoding sequence.

Materials and methods

After design, the modified proinsulin encoding sequence containing a 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 PCR using seven specific primers in multiple sequential PCR reactions. The final amplified fragment was cloned in pGEM-5zf vector by EcoRV enzyme. After sequencing, the modified proinsulin fragment was sub-cloned into pET-26b(+) expression vector by using the NdeI and XhoI enzymes. Finally, expression of the modified proinsulin was evaluated in E. coli BL-21(DE3) by induction with IPTG.

Results

Synthesis and integrity of the modified proinsulin sequence were confirmed by DNA sequencing. Cloning of the modified proinsulin sequence was confirmed by specific PCR amplification and restriction enzyme mapping. In this study, recombinant modified proinsulin protein was expressed up to 40%. Expression of the recombinant protein was confirmed using SDS-PAGE and western blotting.

Conclusions

This modified recombinant proinsulin protein can be used for simple and efficient production of the insulin glargine analogue without any impurity following thrombin treatment within the nickel-chromatography column.

Language:
English
Published:
Research in Molecular Medicine, Volume:9 Issue: 3, Aug 2021
Pages:
189 to 195
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