Preparation of a new thermo‐responsive adsorbent with maltose as a ligand and its application to affinity precipitation

Abstract

A thermo-responsive polymer on which maltose was covalently immobilized as an affinity ligand was newly synthesized for purification of thermolabile proteins from the crude solution by affinity precipitation. Among the thermo-responsive polymers synthesized as carriers for adsorbent, poly(N-acryloylpiperidine)–cysteamine (pAP) has a lower critical solution temperature (LCST) of around 4°C, at which its solubility exhibits a sharp change. Adsorbent for affinity precipitation was prepared by combining pAP with maltose using trimethylamine–borane as a reducing reagent. This adsorbent (pAPM) obtained showed a good solubility response: pAPM in the basal buffer (pH 7.0) became soluble below 4°C and was completely insoluble above 8°C. The affinity precipitation method using pAPM consisted of the following four steps: adsorption at 4°C, precipitation of the complex at 10°C, desorption by adding the desorption reagent at 4°C, and recovery of a target protein at 10°C. In the affinity precipitation of Con A from the crude extract of jack bean meal, 82% of Con A added was recovered with 80% purity by addition of 0.2 M methyl-α-D-mannopyranoside as a desorption reagent. In the repeated purification of Con A from the crude extract, pAPM could be satisfactorily reused without decrease in the affinity performance. Moreover, when pAPM was used for the purification of thermolabile α-glucosidase from the cell-free extract of Saccharomyces cerevisiae, 68% of total activity added was recovered and the specific activity per amount of protein of the purified solution was enhanced 206-fold higher than that of the cell-free extract without thermal deactivation of the enzyme. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 568–579, 1998.