Novel column-based protein refolding strategy using dye-ligand affinity chromatography based on macroporous biomaterial

A novel column-based chromatographic protein refolding strategy was developed using dye-ligand affinity chromatography (DLAC) based on macroporous biomaterial. Chitosan–silica (CS–silica) biomaterial with macroporous surface was used as the supporting matrix for the preparation of the DLAC material. The dye-ligand Cibacron Blue F3GA (CBF) was selected as affinity handle and could be covalently immobilized to form dye-ligand affinity adsorbent (CBF–CS–silica) using the reactivity of NH₂ on CS–silica biomaterial. After the model protein catalase was denatured with 6 mol/L urea, the denaturant could be rapidly removed and catalase could be successfully refolded as facilitated by the adsorption of CBF–CS–silica. The urea denaturation process and the elute condition for the chromatographic refolding were optimized by measuring tryptophan fluorescence and activity of catalase. The refolding performance of the proposed DLAC was compared with dilution refolding. The protein concentration during the proposed chromatographic refolding increased by a factor of 20 without reducing the yield achieved as compared to dilution refolding. The column-based protein refolding strategy based on dye-ligand affinity chromatography with porous biomaterial being matrix possessed potential in chromatographic refolding of protein.