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Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of GK0767, the copper-containing nitrite reductase from Geobacillus kaustophilus

Fukuda, Yota*; Tamada, Taro; Takami, Hideto*; Suzuki, Shinichiro*; Inoue, Tsuyoshi*; Nojiri, Masaki

The soluble region (residues 32-354) of GK0767, a copper-containing nitrite reductase from the thermophilic Gram-positive bacterium ${it Geobacillus kaustophilus}$ HTA426, has been cloned and overexpressed in ${it Escherichia coli}$. The purified recombinant protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected and processed to a maximum resolution of 1.3 ${AA}$. The crystals belonged to space group ${it R}$3, with unit-cell parameters ${it a}$ = ${it b}$ = 115.1, ${it c}$ = 87.5 ${AA}$. Preliminary studies and molecular-replacement calculations reveal the presence of one subunit of the homotrimeric structure in the asymmetric unit; this corresponds to a V$$_{M}$$ value of 3.14 ${AA}$ $$^{3}$$ Da$$^{-1}$$.

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Category:Biochemical Research Methods

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