Kinetic locking‐on and auxiliary tactics for bioaffinity purification of NADP + ‐dependent dehydrogenases using N 6 ‐linked immobilized NADP + derivatives: Studies with mammalian and microbial glutamate dehydrogenases

• Published in: Biotechnology and bioengineering 2003-02, Vol.81 (3), p.356-369
• Main Authors: McMahon, Mary, Tynan, Julie, Mulcahy, Patricia
• Format: Article
• Language: English
• ISSN: 0006-3592; 1097-0290
• 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 μmol/min/mg; purification factor, 83.7; yield 88%). The potential for development of analogous bioaffinity systems for other NADP + ‐dependent dehydrogenases is also discussed. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 356–369, 2003.