Reproducible Formation of Insulin Superstructures: Amyloid-Like Fibrils, Spherulites, and Particulates

Camilla Thorlaksen; Martin Busch Neergaard; Minna Groenning; Vito Foderà

doi:10.1007/978-1-0716-2597-2_20

Reproducible Formation of Insulin Superstructures: Amyloid-Like Fibrils, Spherulites, and Particulates

Camilla Thorlaksen^*, Martin Busch Neergaard, Minna Groenning, Vito Foderà

^*Corresponding author for this work

Drug Delivery and Biophysics of Biopharmaceuticals

Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review

2 Citations (Scopus)

Abstract

Inducing protein aggregation in vitro under various formulation and stress conditions may lead to an increased understanding of the different association routes a protein can undergo. However, a range of factors can affect the aggregation process, often leading to heterogenous samples and experimental irreproducibility between labs. Here, we present detailed methods to reproducibly form homogenous samples of superstructures: amyloid-like fibrils, spherulites, and particulates from human insulin. We discuss pitfalls and good practice in the lab, with the aim of creating awareness on the potential sources of artefacts for protein stability and aggregation studies.

Original language	English
Title of host publication	Protein Aggregation : Methods in Molecular Biology
Editors	A. Stanisław Cieplak
Publisher	Humana Press
Publication date	2023
Pages	297-309
Chapter	20
ISBN (Print)	978-1-0716-2596-5
ISBN (Electronic)	978-1-0716-2597-2
DOIs	https://doi.org/10.1007/978-1-0716-2597-2_20
Publication status	Published - 2023

Series	Methods in Molecular Biology
Volume	2551
ISSN	1064-3745

Bibliographical note

Funding Information:
The work was funded, and all protein material was provided by Novo Nordisk A/S. For use of the TEM, we acknowledge the Core Facility for Integrated Microscopy, Faculty of Health and Medical Sciences, University of Copenhagen. For use of the MFI image evaluation software, we acknowledge Jesper Søndergaard Marino from Novo Nordisk A/S. Illustrations presented in the chapter were created with Biorender.com. V.F. also acknowledges the VIL-LUM FONDEN for the Villum Young Investigator Grant “Protein Superstructures as Smart Biomaterials (ProSmart)” 2018–2023 (project number: 19175). The authors acknowledge Marco van de Weert (University of Copenhagen) for inspiring discussions on reproducibility in protein stability and aggregation studies.

Publisher Copyright:
© 2023, Springer Science+Business Media, LLC, part of Springer Nature.

Keywords

Aggregation
Amyloid-like fibrils
Insulin
Micro-Flow Imaging
Particulates
Spherulites
Superstructures
Transmission electron microscopy

Access to Document

10.1007/978-1-0716-2597-2_20

Cite this

Thorlaksen, C., Neergaard, M. B., Groenning, M., & Foderà, V. (2023). Reproducible Formation of Insulin Superstructures: Amyloid-Like Fibrils, Spherulites, and Particulates. In A. Stanisław Cieplak (Ed.), Protein Aggregation: Methods in Molecular Biology (pp. 297-309). Humana Press. Methods in Molecular Biology Vol. 2551 https://doi.org/10.1007/978-1-0716-2597-2_20

Reproducible Formation of Insulin Superstructures : Amyloid-Like Fibrils, Spherulites, and Particulates. / Thorlaksen, Camilla; Neergaard, Martin Busch; Groenning, Minna; Foderà, Vito.

Protein Aggregation: Methods in Molecular Biology. ed. / A. Stanisław Cieplak. Humana Press, 2023. p. 297-309 (Methods in Molecular Biology, Vol. 2551).

Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review

@inbook{abcf13b4a3e040848ce61da85b16c621,

title = "Reproducible Formation of Insulin Superstructures: Amyloid-Like Fibrils, Spherulites, and Particulates",

abstract = "Inducing protein aggregation in vitro under various formulation and stress conditions may lead to an increased understanding of the different association routes a protein can undergo. However, a range of factors can affect the aggregation process, often leading to heterogenous samples and experimental irreproducibility between labs. Here, we present detailed methods to reproducibly form homogenous samples of superstructures: amyloid-like fibrils, spherulites, and particulates from human insulin. We discuss pitfalls and good practice in the lab, with the aim of creating awareness on the potential sources of artefacts for protein stability and aggregation studies.",

keywords = "Aggregation, Amyloid-like fibrils, Insulin, Micro-Flow Imaging, Particulates, Spherulites, Superstructures, Transmission electron microscopy",

author = "Camilla Thorlaksen and Neergaard, {Martin Busch} and Minna Groenning and Vito Foder{\`a}",

note = "Funding Information: The work was funded, and all protein material was provided by Novo Nordisk A/S. For use of the TEM, we acknowledge the Core Facility for Integrated Microscopy, Faculty of Health and Medical Sciences, University of Copenhagen. For use of the MFI image evaluation software, we acknowledge Jesper S{\o}ndergaard Marino from Novo Nordisk A/S. Illustrations presented in the chapter were created with Biorender.com. V.F. also acknowledges the VIL-LUM FONDEN for the Villum Young Investigator Grant “Protein Superstructures as Smart Biomaterials (ProSmart)” 2018–2023 (project number: 19175). The authors acknowledge Marco van de Weert (University of Copenhagen) for inspiring discussions on reproducibility in protein stability and aggregation studies. Publisher Copyright: {\textcopyright} 2023, Springer Science+Business Media, LLC, part of Springer Nature.",

year = "2023",

doi = "10.1007/978-1-0716-2597-2_20",

language = "English",

isbn = "978-1-0716-2596-5",

series = "Methods in Molecular Biology",

publisher = "Humana Press",

pages = "297--309",

editor = "{Stanis{\l}aw Cieplak}, A.",

booktitle = "Protein Aggregation",

address = "United States",

}

TY - CHAP

T1 - Reproducible Formation of Insulin Superstructures

T2 - Amyloid-Like Fibrils, Spherulites, and Particulates

AU - Thorlaksen, Camilla

AU - Neergaard, Martin Busch

AU - Groenning, Minna

AU - Foderà, Vito

N1 - Funding Information: The work was funded, and all protein material was provided by Novo Nordisk A/S. For use of the TEM, we acknowledge the Core Facility for Integrated Microscopy, Faculty of Health and Medical Sciences, University of Copenhagen. For use of the MFI image evaluation software, we acknowledge Jesper Søndergaard Marino from Novo Nordisk A/S. Illustrations presented in the chapter were created with Biorender.com. V.F. also acknowledges the VIL-LUM FONDEN for the Villum Young Investigator Grant “Protein Superstructures as Smart Biomaterials (ProSmart)” 2018–2023 (project number: 19175). The authors acknowledge Marco van de Weert (University of Copenhagen) for inspiring discussions on reproducibility in protein stability and aggregation studies. Publisher Copyright: © 2023, Springer Science+Business Media, LLC, part of Springer Nature.

PY - 2023

Y1 - 2023

N2 - Inducing protein aggregation in vitro under various formulation and stress conditions may lead to an increased understanding of the different association routes a protein can undergo. However, a range of factors can affect the aggregation process, often leading to heterogenous samples and experimental irreproducibility between labs. Here, we present detailed methods to reproducibly form homogenous samples of superstructures: amyloid-like fibrils, spherulites, and particulates from human insulin. We discuss pitfalls and good practice in the lab, with the aim of creating awareness on the potential sources of artefacts for protein stability and aggregation studies.

AB - Inducing protein aggregation in vitro under various formulation and stress conditions may lead to an increased understanding of the different association routes a protein can undergo. However, a range of factors can affect the aggregation process, often leading to heterogenous samples and experimental irreproducibility between labs. Here, we present detailed methods to reproducibly form homogenous samples of superstructures: amyloid-like fibrils, spherulites, and particulates from human insulin. We discuss pitfalls and good practice in the lab, with the aim of creating awareness on the potential sources of artefacts for protein stability and aggregation studies.

KW - Aggregation

KW - Amyloid-like fibrils

KW - Insulin

KW - Micro-Flow Imaging

KW - Particulates

KW - Spherulites

KW - Superstructures

KW - Transmission electron microscopy

U2 - 10.1007/978-1-0716-2597-2_20

DO - 10.1007/978-1-0716-2597-2_20

M3 - Book chapter

C2 - 36310211

AN - SCOPUS:85141005126

SN - 978-1-0716-2596-5

T3 - Methods in Molecular Biology

SP - 297

EP - 309

BT - Protein Aggregation

A2 - Stanisław Cieplak, A.

PB - Humana Press

ER -