Expression, purification and characterisation of large quantities of recombinant human IAPP for mechanistic studies

Martin Lundqvist, Diana C. Rodriguez Camargo, Katja Bernfur, Sean Chia, Sara Linse

Research output: Contribution to journalArticlepeer-review

1 Citation (SciVal)

Abstract

Malfunction and amyloid formation of the Islet Amyloid Polypeptide (IAPP) are factors contributing to Type 2 diabetes. Unravelling the mechanism of IAPP aggregate formation may forward our understanding of this process and its effect on pancreatic β-islet cell. Such mechanistic studies require access to sequence homogeneous and highly pure IAPP. Here we present a new facile protocol for the production of pure recombinant human IAPP at relatively high yield. The protocol uses a His-tagged version of the Npro mutant EDDIE, which drives expression to inclusion bodies, from which the peptide is purified using sonication, refolding and auto-cleavage, removal of EDDIE using Ni-NTA chromatography and reverse-phase HPLC. The purified material is used at multiple concentrations in aggregation kinetics measurements monitored by thioflavin-T fluorescence. Global analysis of the data implies a double nucleation aggregation mechanism including both primary and secondary nucleation.

Original languageEnglish
Article number106511
JournalBiophysical Chemistry
Volume269
DOIs
Publication statusPublished - 2021 Feb

Subject classification (UKÄ)

  • Biochemistry and Molecular Biology

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