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

Lundqvist, Martin LU ; Rodriguez Camargo, Diana C. LU ; Bernfur, Katja LU ; Chia, Sean and Linse, Sara LU (2021) In Biophysical Chemistry 269.
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... (More)

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.

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author
; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biophysical Chemistry
volume
269
article number
106511
publisher
Elsevier
external identifiers
  • pmid:33360112
  • scopus:85099234213
ISSN
0301-4622
DOI
10.1016/j.bpc.2020.106511
language
English
LU publication?
yes
id
f25ea37d-bc6d-4869-b50f-eb34c4c23e7b
date added to LUP
2021-01-25 08:44:20
date last changed
2022-08-04 06:56:33
@article{f25ea37d-bc6d-4869-b50f-eb34c4c23e7b,
  abstract     = {{<p>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 N<sup>pro</sup> 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.</p>}},
  author       = {{Lundqvist, Martin and Rodriguez Camargo, Diana C. and Bernfur, Katja and Chia, Sean and Linse, Sara}},
  issn         = {{0301-4622}},
  language     = {{eng}},
  publisher    = {{Elsevier}},
  series       = {{Biophysical Chemistry}},
  title        = {{Expression, purification and characterisation of large quantities of recombinant human IAPP for mechanistic studies}},
  url          = {{http://dx.doi.org/10.1016/j.bpc.2020.106511}},
  doi          = {{10.1016/j.bpc.2020.106511}},
  volume       = {{269}},
  year         = {{2021}},
}