Premium
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
Author(s) -
Teshima Tadanaru,
Kohda Jiro,
Kondo Akihiko,
Yohda Masafumi,
Tamura Ai,
Fukuda Hideki
Publication year - 2000
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp0000095
Subject(s) - thermophile , escherichia coli , thermus , biochemistry , thermus thermophilus , denaturation (fissile materials) , malate dehydrogenase , biology , groel , chaperonin , affinity chromatography , chaperone (clinical) , thermostability , bacteria , strain (injury) , bacillus (shape) , enzyme , chemistry , protein folding , microbiology and biotechnology , gene , anatomy , nuclear chemistry , medicine , genetics , pathology
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 °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 °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.