title: Expression, purification and preliminary crystallographic studies of human glutamate oxaloacetate transaminase 1 (GOT1).
abstract: Glutamate oxaloacetate transaminase (GOT) catalyzes the reversible reaction of l-aspartate and α-ketoglutarate into oxaloacetate and L-glutamate and plays a key role in carbon and nitrogen metabolism in all organisms. In human tissues, GOTs are pyridoxal 5'-phosphate-dependent (PLP) enzymes which exist in cytoplasm and mitochondrial forms, GOT1 and GOT2, respectively. GOT1 expression correlates with the growth of several tumors because cancer cells can utilize the amino acid glutamine to fuel anabolic processes, and therefore, GOT1 represents a new therapeutic target in cancer. In this work, human GOT1 was expressed in Escherichia coli periplasmic space, and purified by a combination of His-tag immobilized metal-ion affinity chromatography and anion exchange chromatography. Optimal activity of the enzyme occurred at a temperature of 37 °C and a pH of 7.5. Cations such as Na(+), K(+) and Mg(2+) slightly inhibited the activity of recombinant human GOT1, while Zn(2+), Mn(2+), Cu(2+), Ni(2+), Co(2+) and Ca(2+) had stronger inhibitory effects. Crystals of human GOT1 were grown using the hanging-drop vapor diffusion method at 4°C with 0.1M Bis-Tris pH 6.0% and 21% (w/v) PEG 3350. The crystals diffracted to 2.99Å resolution and belonged to space group P43212 with the unit cell parameters a = b = 93.4, c = 107.4Å, α = β = γ = 90°.
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China. Electronic address: xpjiang@dlut.edu.cn.
authors.forename: Xiuping
authors.initials: X
authors.lastname: Jiang
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China.
authors.forename: Haiyang
authors.initials: H
authors.lastname: Chang
authors.affiliation: School of Software, Dalian University of Technology, Dalian 116620, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhou
mesh_terms: D024443:Aspartate Aminotransferase, Cytoplasmic / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D024443:Aspartate Aminotransferase, Cytoplasmic
keywords: Aspartate aminotransferase; Cancer metabolism; Expression; Glutamate oxaloacetate transaminase; Glutamine addiction; X-ray crystallography