Escherichia coli chaperonin GroEL with a hexa-histidine affinity tag (His)(6) fused to its C-terminal (GroEL(His)6) was overproduced in E. coli. GroEL-(His)(6) was expressed in a soluble form in E. coli and purified to homogeneity in a single step by nickel chelate resin. GroEL-(His)(6) was found to form a tetradecamer and showed a similar ATPase activity to native GroEL. GroEL-(His)(6) mediated refolding of guanidine hydrochloride-unfolded yeast enolase and reactivation of thermally inactivated yeast enolase to similar extents as native GroEL. The structure and function of GroEL are thus little affected by the fusion of the (His)(6) tag to the C-terminal of GroEL. GroEL-(His)(6) was efficiently immobilized on nickel chelate Cellulofine, and immobilized GroEL-(His)(6) retained a high ability to mediate protein refolding. Therefore, GroEL-(His)(6) is advantageous in the application of chaperonin to protein refolding systems.