人谷氨酸草酰乙酸转氨酶1（GOT1）的表达，纯化和初步晶体学研究。,Protein Expression and Purification

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人谷氨酸草酰乙酸转氨酶1（GOT1）的表达，纯化和初步晶体学研究。
Protein Expression and Purification ( IF 1.4 ) Pub Date : 2015-05-25 , DOI: 10.1016/j.pep.2015.05.010
Xiuping Jiang ₁ , Haiyang Chang ₁ , Yong Zhou ₂

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谷氨酸草酰乙酸转氨酶（GOT）催化L-天冬氨酸和α-酮戊二酸可逆反应生成草酰乙酸和L-谷氨酸，并在所有生物的碳和氮代谢中起关键作用。在人体组织中，GOT是吡咯醛5'-磷酸盐依赖性（PLP）酶，分别以细胞质和线粒体形式GOT1和GOT2存在。GOT1表达与几种肿瘤的生长相关，因为癌细胞可以利用氨基酸谷氨酰胺来促进合成代谢过程，因此GOT1代表了癌症的新治疗靶点。在这项工作中，人GOT1在大肠杆菌周质空间中表达，并通过结合His-tag的固定化金属离子亲和色谱法和阴离子交换色谱法纯化。该酶的最佳活性发生在温度为37°C和pH值为7.5的情况下。诸如Na（+），K（+）和Mg（2+）之类的阳离子略微抑制了重组人GOT1的活性，而Zn（2 +），Mn（2 +），Cu（2 +），Ni（2+ ），Co（2+）和Ca（2+）具有更强的抑制作用。使用悬滴气相扩散法在0.1°Bis-Tris pH 6.0％和21％（w / v）PEG 3350于4°C下生长人GOT1晶体。该晶体衍射至2.99Å分辨率，属于空间群P43212的晶胞参数a = b = 93.4，c =107.4Å，α=β=γ= 90°。

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

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°.

更新日期：2015-05-21

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