Expression, purification, and characterization of recombinant human glutamine synthetase.
Expression, purification, and characterization of recombinant human glutamine synthetase.
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DOI：
10.1042/bj3280159
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发表时间：
1997-11
期刊：
影响因子：
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通讯作者：
Chad D. Listrom;H. Morizono;Basavapatna S. Rajagopal;Mark T. McCANN;Mendel Tuchman;N. Allewell
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文献类型：
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作者：
Chad D. Listrom;H. Morizono;Basavapatna S. Rajagopal;Mark T. McCANN;Mendel Tuchman;N. Allewell
已针对人谷氨酰胺合成酶 (EC 6.3.1.2) 设计了细菌表达系统，可产生约 60 毫克酶（细菌可溶性蛋白的 20%），并产生约 10% 的酶。每升培养物含有 8 毫克纯化酶。将重组酶纯化 5 倍以达到明显的同质性并进行表征。它的亚基分子质量约为。 45000 达。以氨和l-[G-3H]谷氨酸为底物的放射性测定得到的Vmax值为15.9微摩尔/分钟/毫克，比用羟胺代替氨作为底物的比色测定法(9.9微摩尔/分钟/毫克)得到的Vmax值高40%。对于放射性和分光光度测定，谷氨酸的 Km 值分别为 3.0 mM 和 3.5 mM，ATP 的 Km 值分别为 2.0 mM 和 2. 9 mM。放射性测定中氨的 Km 为 0.15 mM。热失活的中点为 49.7 摄氏度。羟胺、Mg(II) 和 Mg(II)-ATP 可以稳定酶，防止热失活，而 ATP 则促进失活。纯酶可在储存中稳定数月，并为其他研究（包括 X 射线晶体学）提供来源。
A bacterial expression system has been engineered for human glutamine synthetase (EC 6.3.1.2) that produces approximately 60 mg of enzyme (20% of the bacterial soluble protein) and yields approx. 8 mg of purified enzyme per litre of culture. The recombinant enzyme was purified 5-fold to apparent homogeneity and characterized. It has a subunit molecular mass of approx. 45000 Da. The Vmax value obtained using a radioactive assay with ammonia and l-[G-3H]glutamic acid as substrates was 15.9 micromol/min per mg, 40% higher than that obtained in the colorimetric assay (9.9 micromol/min per mg) with hydroxylamine replacing ammonia as a substrate. Km values for glutamate were 3.0 mM and 3.5 mM, and for ATP they were 2.0 mM and 2. 9 mM for the radioactive and spectrophotometric assays respectively. The Km for ammonia in the radioactive assay was 0.15 mM. The midpoint of thermal inactivation was 49.7 degrees C. Hydroxylamine, Mg(II) and Mg(II)-ATP stabilized the enzyme against thermal inactivation, whereas ATP promoted inactivation. The pure enzyme is stable for several months in storage and provides a source for additional studies, including X-ray crystallography.