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 | Zendy

Teshima Tadanaru | Zendy; Kohda Jiro | Zendy; Kondo Akihiko | Zendy; Yohda Masafumi | Zendy; Tamura Ai | Zendy; Fukuda Hideki | Zendy

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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.

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