McMahon Mary, Tynan Julie, Mulcahy Patricia
Department of Applied Biology and Chemistry, Institute of Technology, Carlow, Ireland.
Biotechnol Bioeng. 2003 Feb 5;81(3):356-69. doi: 10.1002/bit.10547.
This study is concerned with the development and application of kinetic locking-on and auxiliary tactics for bioaffinity purification of NADP(+)-dependent dehydrogenases, specifically (1) the synthesis and characterization of highly substituted N(6)-linked immobilized NADP(+) derivatives using a rapid solid-phase modular approach; (2) the evaluation of the N(6)-linked immobilized NADP(+) derivatives for use with the kinetic locking-on strategy for bioaffinity purification of NADP(+)-dependent dehydrogenases: Model bioaffinity chromatographic studies with glutamate dehydrogenase from bovine liver (GDH with dual cofactor specificity, EC 1.4.1.3) and glutamate dehydrogenase from Candida utilis (GDH which is NADP(+)-specific, EC 1.4.1.4); (3) the selection of an effective "stripping ligand" for NADP(+)-dehydrogenase bioaffinity purifications using N(6)-linked immobilized NADP(+) derivatives in the locking-on mode; and (4) the application of the developed bioaffinity chromatographic system to the purification of C. utilis GDH from a crude cellular extract. Results confirm that the newly developed N(6)-linked immobilized NADP(+) derivatives are suitable for the one-step bioaffinity purification of NADP(+)-dependent GDH provided that they are used in the locking-on mode, steps are taken to inhibit alkaline phosphatase activity in crude cellular extracts, and 2',5'-ADP is used as the stripping ligand during chromatography. The general principles described here are supported by a specific sample enzyme purification; the purification of C. utilis GDH to electrophoretic homogeneity in a single bioaffinity chromatographic step (specific activity, 9.12 micromol/min/mg; purification factor, 83.7; yield 88%). The potential for development of analogous bioaffinity systems for other NADP(+)-dependent dehydrogenases is also discussed.
本研究关注用于NADP(+)依赖性脱氢酶生物亲和纯化的动力学锁定及辅助策略的开发与应用，具体包括：(1) 使用快速固相模块化方法合成并表征高度取代的N(6)-连接固定化NADP(+)衍生物；(2) 评估N(6)-连接固定化NADP(+)衍生物用于NADP(+)依赖性脱氢酶生物亲和纯化的动力学锁定策略：以牛肝谷氨酸脱氢酶（具有双辅因子特异性的谷氨酸脱氢酶，EC 1.4.1.3）和产朊假丝酵母谷氨酸脱氢酶（NADP(+)特异性的谷氨酸脱氢酶，EC 1.4.1.4）进行模型生物亲和色谱研究；(3) 在锁定模式下使用N(6)-连接固定化NADP(+)衍生物选择用于NADP(+)脱氢酶生物亲和纯化的有效“洗脱配体”；(4) 将开发的生物亲和色谱系统应用于从粗细胞提取物中纯化产朊假丝酵母谷氨酸脱氢酶。结果证实，新开发的N(6)-连接固定化NADP(+)衍生物适用于NADP(+)依赖性谷氨酸脱氢酶的一步生物亲和纯化，前提是它们以锁定模式使用，采取措施抑制粗细胞提取物中的碱性磷酸酶活性，并在色谱过程中使用2',5'-ADP作为洗脱配体。这里描述的一般原则通过特定样品酶的纯化得到支持；在单个生物亲和色谱步骤中将产朊假丝酵母谷氨酸脱氢酶纯化至电泳纯（比活性，9.12 μmol/min/mg；纯化倍数，83.7；产率88%）。还讨论了开发用于其他NADP(+)依赖性脱氢酶的类似生物亲和系统的潜力。
