Expression, purification, and PC1-mediated processing of (H10D, P28K, and K29P)-human proinsulin

Robert B. Mackin, Meredith H. Choquette

Research output: Contribution to journalArticle

14 Scopus citations


Our previous methods for the generation of recombinant human proinsulin were inadequate in terms of reproducibility and yield. In addition, it was difficult to perform structure/function studies on proinsulin because of its tendency to form hexamers. We have developed an improved procedure, which overcomes many of the technical purification problems, and results in a potentially monomeric version of modified proinsulin. Inclusion bodies were prepared using a commercial bacterial lysis solution. The inclusion bodies were solubilized and the fusion protein's affinity tag was removed by chemical cleavage. The polypeptide was then reduced and transferred into a refolding buffer. Following an overnight incubation, only a single form of proinsulin was detected using analytical reversed-phase high-performance liquid chromatography. The refolded (H10D, P28K, and K29P)-human proinsulin (DKP-hPI) was subjected to a final purification step using reversed-phase chromatography. The method is reproducible and produces milligram quantities of purified DKP-hPI from a single liter of bacterial culture. The final product is greater than 95% pure and is suitable for use as a substrate for the propeptide convertase PC1.

Original languageEnglish (US)
Pages (from-to)210-219
Number of pages10
JournalProtein Expression and Purification
Issue number2
StatePublished - Feb 1 2003

All Science Journal Classification (ASJC) codes

  • Biotechnology

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