McMahon Mary, Mulcahy Patricia
Department of Applied Biology and Chemistry, Institute of Technology Carlow, Kilkenny Road, Carlow, Ireland.
Biotechnol Bioeng. 2002 Mar 5;77(5):517-27. doi: 10.1002/bit.10079.
This study describes the development and application of a bioaffinity chromatographic system for the one-step purification of an NADP(+)-dependent secondary alcohol dehydrogenase from the obligate anaerobe, Thermoanaerobacter brockii (TBADH, EC 1.1.1.2). The general approach is based upon improving the selectivity of immobilized cofactor derivatives (general ligand approach to bioaffinity chromatography) through using soluble enzyme-specific substrate analogues in irrigants to promote biospecific adsorption (the kinetic locking-on tactic). Specifically, the following is described: Evaluation of 8'-azo-linked, C(8)-linked, N(1)-linked, and N(6)-linked immobilized NADP(+) derivatives for use with the kinetic locking-on strategy for bioaffinity purification of TBADH; evaluation of 2', 5'-ADP as a stripping ligand for TBADH bioaffinity purifications using an 8'-azo-linked immobilized NADP(+) derivative in the locking-on mode; and application of the developed bioaffinity chromatographic system to the purification of TBADH from a crude cellular extract. Surprizingly, of the four immobilized NADP(+) derivatives investigated, only the 8'-azo-linked immobilized NADP(+) derivative proved effective for TBADH affinity purification when used in conjunction with pyrazole (a competitive inhibitor of TBADH activity) as the locking-on ligand. This is in contrast to other NADP(+)-dependent dehydrogenases where the immobilized N(6)-linked cofactor proved to be suitable. While the one-step purification of TBADH to electrophoretic homogeneity is described in the present study (92% yield), results from the model chromatographic studies point to improvements that could be made to the immobilized cofactor derivative to improve its suitability for TBADH bioaffinity purification and to facilitate future large scale protein purification operations.
本研究描述了一种生物亲和色谱系统的开发与应用，该系统用于从专性厌氧菌布氏嗜热栖热菌中一步纯化NADP(+)依赖性仲醇脱氢酶（TBADH，EC 1.1.1.2）。总体方法是通过在冲洗液中使用可溶性酶特异性底物类似物来促进生物特异性吸附（动力学锁定策略），从而提高固定化辅因子衍生物的选择性（生物亲和色谱的通用配体方法）。具体内容如下：评估8'-偶氮连接、C(8)连接、N(1)连接和N(6)连接的固定化NADP(+)衍生物，用于TBADH生物亲和纯化的动力学锁定策略；评估2', 5'-ADP作为脱附配体，用于在锁定模式下使用8'-偶氮连接的固定化NADP(+)衍生物进行TBADH生物亲和纯化；以及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TBADH。令人惊讶的是，在所研究的四种固定化NADP(+)衍生物中，只有8'-偶氮连接的固定化NADP(+)衍生物在与吡唑（TBADH活性的竞争性抑制剂）作为锁定配体结合使用时，被证明对TBADH亲和纯化有效。这与其他NADP(+)依赖性脱氢酶不同，在其他脱氢酶中，固定化的N(6)连接辅因子被证明是合适的。虽然本研究描述了将TBADH一步纯化为电泳纯（产率92%），但模型色谱研究结果表明，可以对固定化辅因子衍生物进行改进，以提高其对TBADH生物亲和纯化的适用性，并便于未来的大规模蛋白质纯化操作。
