Affinity purification of fusion chaperonin cpn60-(His)_6 from thermophilic bacterium Bacillus strain MS and its use in facilitating protein refolding and preventing heat denaturation

摘要

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)_6was 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 hydrochlorideunfolded pig heart malic dehydrogenase (MDH) and Thermus flavus MDH at 25 and 70 deg 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 deg 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.