Refolding of urea denatured ovalbumin with three phase partitioning generates many conformational variants

•Urea denatured ovalbumin was refolded and showed anti-trypsin activity.•Refolding was done by precipitation with ammonium sulphate and t-butanol.•Different refolding conditions gave rise to different conformational variants.•Refolded ovalbumin was prone to amyloidogenesis. Three phase partitioning is a process in which mixing t-butanol with ammonium sulphate with a protein solution leads to the formation of three phases. Generally, the interfacial protein precipitate (formed between upper t-butanol rich and lower aqueous phase) can be easily dissolved back in aqueous buffers. In case of ovalbumin, this led to a precipitate which was insoluble in aqueous buffers. This precipitate when solubilized with 8M urea and subjected to three phase partitioning under various conditions led to many refolded soluble conformational variants of ovalbumin. One of these showed trypsin inhibitory activity, had marginally higher β-sheet content and had higher surface hydrophobicity (both with respect to native ovalbumin). Scanning electron microscopy and Atomic force microscopy of this preparation showed a thread like structure characteristic of amyloid fibrils. The behaviour of ovalbumin during three phase partitioning makes it a valuable system for gaining further understanding of protein aggregation.