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.
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.
Teshima
T
T
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.
Kohda
J
J
Kondo
A
A
Yohda
M
M
Tamura
A
A
Fukuda
H
H
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
D018834:Chaperonin 60; D017931:DNA Primers; D006639:Histidine; D019791:Guanidine