Expression, purification and preliminary crystallographic studies of human glutamate oxaloacetate transaminase 1 (GOT1)

摘要

Glutamate oxaloacetate transaminase (GOT) catalyzes the reversible reaction of L-aspartate and alpha-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 coil 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 degrees C and a pH of 7.5. Cations such as Na+, K+ and Mg2+ slightly inhibited the activity of recombinant human GOT1, while Zn2+, Mn2+, Cu2+, Ni2+, Co2+ and Ca2+ had stronger inhibitory effects. Crystals of human GOT1 were grown using the hanging-drop vapor diffusion method at 4 degrees C with 0.1 M Bis-Tris pH 6.0% and 21% (w/v) PEG 3350. The crystals diffracted to 2.99 angstrom resolution and belonged to space group P4(3)2(1)2 with the unit cell parameters a = b = 93.4, c = 107.4 angstrom, alpha = beta = gamma = 90 degrees. (C) 2015 Elsevier Inc. All rights reserved.