PREPARATION OF A NEW THERMORESPONSIVE ADSORBENT WITH MALTOSE AS A LIGAND AND ITS APPLICATION TO AFFINITY PRECIPITATION

A thermo-responsive polymer on which maltose was covalently immobilize d as an affinity ligand was newly synthesized for purification of ther molabile proteins from the crude solution by affinity precipitation. A mong the thermo-responsive polymers synthesized as carriers for adsorb ent, poly(N-acryloylpiperidine)cysteamine (pAP) has a lower critical s olution temperature (LCST) of around 4 degrees C, at which its solubil ity 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 solub ility response: pAPM in the basal buffer (pH 7.0) became soluble below 4 degrees C and was completely insoluble above 8 degrees C. The affin ity precipitation method using pAPM consisted of the following four st eps: adsorption at 4 degrees C, precipitation of the complex at 10 deg rees C, desorption by adding the desorption reagent at 4 degrees C, an d recovery of a target protein at 10 degrees C. In the affinity precip itation 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-alph a-D-mannopyranoside as a desorption reagent. In the repeated purificat ion of Con A from the crude extract, pAPM could be satisfactorily reus ed without decrease in the affinity performance. Moreover, when pAPM w as used for the purification of thermolabile cr-glucosidase from the c ell-free extract of Saccharomyces cerevisiae, 68% of total activity ad ded was recovered and the specific activity per amount of protein of t he purified solution was enhanced 206-fold higher than that of the cel l-free extract without thermal deactivation of the enzyme. (C) 1998 Jo hn Wiley & Sons, Inc.