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 degrees 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 degrees C and was completely insoluble above 8 degrees C. The affinity precipitation method using pAPM consisted of the following four steps: adsorption at 4 degrees C, precipitation of the complex at 10 degrees C, desorption by adding the desorption reagent at 4 degrees C, and recovery of a target protein at 10 degrees 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-alpha-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 alpha-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.

Smart hybrid materials by conjugation of responsive polymers to biomacromolecules

“…Temperature-responsive poly(N-acryloylpiperidine) (PAP) can be functionalized via its amino end group with a disaccharide, maltose, as an affinity ligand. The resulting PAP-maltose conjugate was used to efficiently purify a thermolabile protein, Con A, from the crude extract of jack bean meal by affinity precipitation 160 . Similarly, lectin receptors (Con A or wheat germ lectin (WGL)) were conjugated to a PNIPAM hydrogel, and used as affinity ligands.…”

Novel adsorbents and approaches for nutraceutical separation

“…Cycling below and above the LCST of the temperature-responsive polymer induces reversible precipitation/ solubilization of the bioconjugates in the aqueous solution, thereby allowing separation of the bioconjugate and the target molecule. Proteins such as a-chymotrypsin (Kim & Park, 1998) and a-glucosidase (Hoshino et al, 1998) were purified using this method. In addition, temperature-dependent isolation of lactate dehydrogenase from porcine muscle was achieved using a dye-affinity agarose modified by physically adsorbing the smart polymer poly-N-vinylcaprolactam onto the surface.…”

Functional Glyco-Affinity Precipitation/Capturing for Enhanced Affinity Proteomics

“…The cloud point as a function of polymer concentration often exhibits a minimum temperature called the lower critical solution temperature (LCST). Synthetic polymers, Nisopropylacrylamide, N, N-diethylacrylamide, N-vinylcaprolactam, N-cyclopropylacrylamide and poly(N-acryloylpiperidine) with different LCST have been intensively investigated in the past decade (Schild, 1992;Hoshino et al, 1998;Idziak et al, 1999;Lozinsky et al, 2000). Specifically, homopolymers, poly (N-isopropylacrylamide) (polyNIPAM) has received much attention due to its sharp phase transition at 32 °C (Schild 1992).…”

“…An early successful example of thermal-responsive glyco-affinity precipitation was demonstrated by Hoshino et, al. by using synthetic thermo-responsive polymer of poly(Nacryloylpiperidine)-cysteamine (poly-AP) on which maltose as an affinity ligand was covalently immobilized (Hoshino et al, 1998). The adsorbent for affinity precipitation was prepared by combining pAP with maltose using trimethylamine-borane as a reducing reagent and showed a good solubility response: poly-APM in the basal buffer (pH 7.0) became soluble below 4°C and was completely insoluble above 8°C.…”