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

Part of the Methods in Molecular Biology book series (MIMB,volume 2551)


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.

Key words

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

Authors Minna Groenning and Vito Foderà share the last authorship of the manuscript.

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  1. Fodera V, Zaccone A, Lattuada M et al (2013) Electrostatics controls the formation of amyloid superstructures in protein aggregation. Phys Rev Lett 111(10):1–5.

    CrossRef  CAS  Google Scholar 

  2. Wälchli R, Vermeire P-J, Massant J et al (2019) Accelerated aggregation studies of monoclonal antibodies: considerations for storage stability. J Pharm Sci 109(1):595–602.

    CrossRef  CAS  PubMed  Google Scholar 

  3. Rosenberg AS, Worobec AS (2004) A risk-based approach to immunogenicity concerns of therapeutic protein products, part 2: considering host-specific and product-specific factors impacting immunogenicity. BioPharm Int 17(12):34–42

    Google Scholar 

  4. Hermeling S, Schellekens H, Maas C et al (2006) Antibody response to aggregated human interferon alpha2b in wild-type and transgenic immune tolerant mice depends on type and level of aggregation. J Pharm Sci 95(5):1084–1096.

    CrossRef  CAS  PubMed  Google Scholar 

  5. Vetri V, Fodera V (2015) The route to protein aggregate superstructures: particulates and amyloid-like spherulites. FEBS Lett 589(19A):2448–2463.

    CrossRef  CAS  PubMed  Google Scholar 

  6. Vestergaard B, Groenning M, Roessle M et al (2007) A helical structural nucleus is the primary elongating unit of insulin amyloid fibrils. PLoS Biol 5(5):1089–1097.

    CrossRef  CAS  Google Scholar 

  7. Jansen R, Dzwolak W, Winter R (2005) Amyloidogenic self-assembly of insulin aggregates probed by high resolution atomic force microscopy. Biophys J 88(2):1344–1353.

    CrossRef  CAS  PubMed  Google Scholar 

  8. Ahmad A, Uversky VN, Hong D et al (2005) Early events in the fibrillation of monomeric insulin. J Biol Chem 280(52):42669–42675.

    CrossRef  CAS  PubMed  Google Scholar 

  9. Fodera V, Donald AM (2010) Tracking the heterogeneous distribution of amyloid spherulites and their population balance with free fibrils. Eur Phys J E 33(4):273–282.

    CrossRef  CAS  PubMed  Google Scholar 

  10. De Luca G, Galparsoro DF, Sancataldo G et al (2020) Probing ensemble polymorphism and single aggregate structural heterogeneity in insulin amyloid self-assembly. J Coll Int Sci 574(1):229–240.

    CrossRef  CAS  Google Scholar 

  11. Krebs MRH, Macphee CE, Miller AF et al (2004) The formation of spherulites by amyloid fibrils of bovine insulin. Proc Natl Acad Sci USA 101(40):14420–14424.

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  12. Fodera V, Vetri V, Wind TS et al (2014) Observation of the early structural changes leading to the formation of protein superstructures. J Phys Chem Lett 5(18):3254–3258.

    CrossRef  CAS  PubMed  Google Scholar 

  13. Krebs MRH, Devlin GL, Donald AM (2007) Protein particulates: another generic form of protein aggregation? Biophys J 92(4):1336–1342.

    CrossRef  CAS  PubMed  Google Scholar 

  14. Vetri V, D’amico M, Fodera V et al (2011) Bovine serum albumin protofibril-like aggregates formation: solo but not simple mechanism. Arch Biochem Biophys 508(1):13–24.

    CrossRef  CAS  PubMed  Google Scholar 

  15. Pedersen MN, Fodera V, Horvath I et al (2015) Direct correlation between ligand-induced alpha-synuclein oligomers and amyloid-like fibril growth. Sci Rep 5(10422):1–11.

    CrossRef  Google Scholar 

  16. Heijna MCR, Theelen MJ, Van Enckevort WJP et al (2007) Spherulitic growth of hen egg-white lysozyme crystals. J Phys Chem B 111(7):1567–1573.

    CrossRef  CAS  PubMed  Google Scholar 

  17. Jiang Y, Shi K, Xia D et al (2011) Protein spherulites for sustained release of interferon: preparation, characterization and in vivo evaluation. J Pharm Sci 100(5):1913–1922.

    CrossRef  CAS  PubMed  Google Scholar 

  18. Lambrecht MA, Jansens KJA, Rombouts I et al (2019) Conditions governing food protein amyloid fibril formation. Part II: milk and legume proteins. Com Rev Food Sci Food Safe 18(1):1277–1291.

    CrossRef  CAS  Google Scholar 

  19. Scheidt T, Łapińska U, Kumita JR et al (2019) Secondary nucleation and elongation occur at different sites on Alzheimer’s amyloid-β aggregates. Sci Adv 5(4):1–9.

    CrossRef  CAS  Google Scholar 

  20. Krebs MRH, Domike KR, Donald AM (2009) Protein aggregation: more than just fibrils. Biochem Soc Trans 37(4):682–686.

    CrossRef  CAS  PubMed  Google Scholar 

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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 V.F. also acknowledges the VILLUM 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.

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Correspondence to Camilla Thorlaksen or Vito Foderà .

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Thorlaksen, C., Neergaard, M.B., Groenning, M., Foderà, V. (2023). Reproducible Formation of Insulin Superstructures: Amyloid-Like Fibrils, Spherulites, and Particulates. In: Cieplak, A.S. (eds) Protein Aggregation. Methods in Molecular Biology, vol 2551. Humana, New York, NY.

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