title: Cloning, overexpression, purification and crystallization of malate dehydrogenase from Thermus thermophilus.
abstract: Malate dehydrogenase (MDH) has been used as a conjugate for enzyme immunoassay of a wide variety of compounds, such as drugs of abuse, drugs used in repetitive therapeutic application and hormones. In consideration of the various biotechnological applications of MDH, investigations of MDH from Thermus thermophilus were carried out to further understand the properties of this enzyme. The DNA fragment containing the open reading frame of mdh was amplified from the genomic DNA of T. thermophilus and cloned into the expression vector pET21b(+). The protein was expressed in a soluble form in Escherichia coli strain BL21(DE3). Homogeneous protein was obtained using a three-step procedure consisting of thermal treatment, Ni(2+)-chelating chromatography and size-exclusion chromatography. The purified MDH was crystallized and the crystals diffracted to a resolution of 1.80 Å on the BL13C1 beamline of the National Synchrotron Radiation Research Center (NSRRC), Taiwan. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 71.3, b = 86.1, c = 118.2 Å. The unit-cell volume of the crystal is compatible with the presence of two monomers in the asymmetric unit, with a corresponding Matthews coefficient VM of 2.52 Å(3) Da(-1) and a solvent content of 51.2%. The crystal structure of MDH has been solved by molecular replacement and is currently under refinement.
authors.affiliation: Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan.
authors.forename: Yu-Yung
authors.initials: YY
authors.lastname: Chang
authors.forename: Chih-Hung
authors.initials: CH
authors.lastname: Hung
authors.forename: Tzann-Shun
authors.initials: TS
authors.lastname: Hwang
authors.forename: Chun-Hua
authors.initials: CH
authors.lastname: Hsu
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification*; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D008291:Malate Dehydrogenase
keywords: TCA cycle; Thermus thermophilus; coupling assays; malate dehydrogenase