The cpn60 gene from Bacillus strain MS, which is highly homologous to Bacillus stearothermophilus, was cloned. Cpn60 with a hexahistidine affinity tag (His)(6) fused to its C-terminus (cpn60-(His)(6)) was overproduced in Escherichia coli. Cpn60-(His)(6) was expressed in a soluble form in E. coli. and purified to homogeneity in a single step by nickel chelate affinity chromatography. Cpn60-(His)(6) formed a tetradecamer and had ATPase activity. Cpn60-(His)(6) mediated refolding of guanidine hydrochloride unfolded pig heart malic dehydrogenase (MDH) and Thermus flavus MDH at 25 and 70 degrees C, respectively, in an ATP-dependent manner. In addition, cpn60-(His)(6) prevented heat denaturation of pig heart MDH and T. flavus MDH at 30 and 80 degrees C, respectively, in an ATP-dependent manner. Therefore, cpn60-(His)(6) facilitates protein refolding and prevents heat denaturation of proteins across a wide temperature range.