Partial denaturation of recombinant protein for affinity purification

Abstract

In this study, we investigate the absence of binding between the 6-His-tagged human transthyretin–green fluorescent protein fusion and the affinity sorbent. Also, the proposal that a partial conformational change makes the 6-His sequence sterically inaccessible and prevents its incorporation into the nickel ion coordination sphere is confirmed. After prolonged incubation (4 weeks) in the presence of imidazole, the protein almost completely lost its capability of binding with Ni-agarose. The treatment of the unbound protein fraction with 0.5 M guanidine hydrochloride recovered this capability. Notably, the recovered protein was in regular conformation and had no signs of denaturation. This approach of partial denaturation resulting in the melted globule can be used for the affinity purification of other recombinant proteins synthesized in bacterial systems, when they are incapable of binding with affinity sorbents or can be solubilized only in the presence of denaturing agents.