Recovery and cryopreservation of insulin amyloid using ionic liquids

Abstract

Amyloid aggregates containing a high content of β-sheet structure are one of several water-insoluble protein aggregates. In this study, we assessed the use of aqueous ionic liquid (IL) solutions as part of a simple preservation/refolding technique for amyloid aggregates. We investigated the dissolution and refolding of cryopreserved bovine insulin amyloid in concentrated aqueous solutions (X = 20 mol%IL) of six different ionic liquids: three 1-butyl-3-methylimidazolium-based ILs ([bmim][X]; X = SCN, NO3, or Cl), 1-ethyl-3-methylimidazolium nitrate ([emim][NO3]), ethylammonium nitrate (EAN), and propylammonium nitrate (PAN). Dimethyl sulfoxide (DMSO) was used as the reference standard for spectroscopy. The six ILs all exhibited the ability to dissolve insulin amyloid, with imidazolium-based ILs exhibiting increased dissolution capacity compared with ammonium-based ILs or DMSO. Remarkably, >80% of the secondary structure of the folded insulin monomer was recovered from insulin amyloid dissolved in aqueous IL solutions following cryopreservation and IL removal by dialysis. These results indicate that concentrated aqueous IL solutions have potential as a new and simple agent for the dissolution, cryopreservation, and refolding of amyloid aggregates.