GroES, the co-chaperonin of Escherichia coli, was overproduced and purified using the maltose binding protein (MBP) fusion vector. The MBP-GroES fusion protein was expressed in a soluble form in E. coli and purified to homogeneity using amylose resin in a single step. The MBP-GroES fusion protein was found to form a heptamer, reduce the ATPase activity of chaperonin GroEL and stimulate GroEL-mediated protein refolding, to a similar extent as the wild-type GroES. Furthermore, the MBP-GroES fusion protein bound GroEL, and this complex was recovered easily from the refolding mixture by amylose resin. These results suggest that the combination of the MBP-GroES fusion protein and GroEL is effective for the construction of a repeated refolding system of proteins.