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

Robert B. Mackin, Meredith H. Choquette

Research output: Contribution to journalArticlepeer-review

16 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 2003
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology


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