Expression, purification and characterisation of large quantities of recombinant human IAPP for mechanistic studies
(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
- Lundqvist, Martin LU ; Rodriguez Camargo, Diana C. LU ; Bernfur, Katja LU ; Chia, Sean and Linse, Sara LU
- organization
- publishing date
- 2021-02
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Biophysical Chemistry
- volume
- 269
- article number
- 106511
- publisher
- Elsevier
- external identifiers
-
- scopus:85099234213
- pmid:33360112
- 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
- 2024-06-13 06:07:52
@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}},
}