Soluble Expression and Purification of Human Proinsulin in Escherichia coli.

Abstract:

The human proinsulin gene（PI） was cloned by PCR using specific primers designed according to the PI gene sequcence in NCBI and codon in Escherichia coli. The expression plasmid pET32-PI was constructed to express fusion protein of PI in E. coli BL21（DE3）. The recombinant PI protein in E. coli exhibited the best expression at an optimized condition of 30 ℃ and 0.6 mmol/L Isopropyl β-D-Thiogalactoside（IPTG）. The results of SDS-PAGE and Western blot analysis indicated that the PI protein was successfully expressed at the same molecular weight as the known PI protein. After the Fed-batch fermentation and centrifugation，this protein was purified to homogeneity using the Ni-NTA affinity chromatography and ion exchange chromatography，respectively. Proinsulin was converted to mature insulin through the digestion of enterokinase and carboxypeptidase. The digested product was purified by Ni-NTA affinity chromatography. The enzyme activity of recombinant insulin was 92 μlU/mL and the product was proved to possess the activity of human insulin.