High-performance hydrophobic interaction chromatography as a tool for protein refolding

Abstract

A method for the refolding of previously unfolded proteins with a concentrated solution of denaturing agent is presented, involving the use of high-performance hydrophobic interaction chromatography (HPHIC) to separate the denaturing agent completely from the unfolded protein and to provide a suitable environment for its refolding. The retention, peak shape and peak height in HPHIC and size-exclusion chromatography, UV spectra, circular dichroic spectra and bioactivity were used to test the possibility and the completeness of the protein refolding. The proposed method permits the extracted solution from Escherichia coli cells to be injected directly into the HPHIC column and, at the same time, the refolding and purification of the protein to be effected. The renaturation and purification of recombinant human interferon — γ from E. coli cells is one example of the application of the method in biotechnology.