title: 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.
abstract: 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.
authors.affiliation: Divisions of Bioscience and Molecular Science, The Graduate School of Science and Technology, and Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan, Divis.
authors.forename: T
authors.initials: T
authors.lastname: Teshima
authors.forename: J
authors.initials: J
authors.lastname: Kohda
authors.forename: A
authors.initials: A
authors.lastname: Kondo
authors.forename: M
authors.initials: M
authors.lastname: Yohda
authors.forename: A
authors.initials: A
authors.lastname: Tamura
authors.forename: H
authors.initials: H
authors.lastname: Fukuda
mesh_terms: D001407:Bacillus / Q000737:chemistry*; D001483:Base Sequence; D018834:Chaperonin 60 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D017931:DNA Primers; D019791:Guanidine / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D006358:Hot Temperature; D011489:Protein Denaturation; D017510:Protein Folding
chemical_list: D018834:Chaperonin 60; D017931:DNA Primers; D006639:Histidine; D019791:Guanidine