Purification of aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli by dye-ligand chromatography.
Purification of aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli by dye-ligand chromatography.
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通过染料-配体色谱法从大肠杆菌中纯化天冬氨酸酶和天冬氨酸激酶-高丝氨酸脱氢酶 I。
DOI：
10.1016/0003-2697(85)90280-5
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发表时间：
1985
影响因子：
2.9
通讯作者：
Viola,RE
中科院分区：
文献类型：
--
作者：
Karsten,WE;Hunsley,JR;Viola,RE
报道了大肠杆菌中L-丝氨酸蛋白酶和肌酸激酶-高丝氨酸脱氢酶I的纯化方法。在这些纯化中，将市售染料基质上的染料-配体色谱法作为关键步骤并入。红色A-琼脂糖对L-天冬氨酸酶具有高亲和力，然后用1 mm L-天冬氨酸将其洗脱为均匀的蛋白质级分。绿色A-琼脂糖显示出对双功能酶胰蛋白激酶-高丝氨酸脱氢酶I的高结合亲和力。通过用NADP+洗脱，随后与NADP和半胱氨酸形成三元复合物（高丝氨酸脱氢酶活性的良好竞争性抑制剂），并在绿色A-琼脂糖上再层析来完成纯化。每种纯化的酶的最终比活性等于或超过先前报道的值，所获得的酶的总产率显著更高，并且发现这些改进的纯化方案更适合于按比例放大以生产大量纯化的酶。
Improved purification schemes are reported for the enzymes l-aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli. Dye-ligand chromatography on commercially available dye matrices are incorporated as key steps in these purifications. Red A-agarose has a high affinity for l-aspartase, which is then eluted as a homogeneous protein fraction with 1 mm l-aspartic acid. Green A-agarose shows a high binding affinity for the bifunctional enzyme aspartokinase-homoserine dehydrogenase I. Purification is accomplished by elution with NADP+, followed by formation of a ternary complex with NADP and cysteine, a good competitive inhibitor of the homoserine dehydrogenase activity, and rechromatography on Green A-agarose. The final specific activity of each purified enzyme equaled or exceed previously reported values, the overall yield of enzymes obtained was significantly higher, and these improved purification schemes were found to be more amenable to being scaled up for the production of large quantities of purified enzyme.