Preparation of a new thermo-responsive adsorbent with maltose as a ligand and its application to affinity precipitation.
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.
Hoshino
K
K
Department of Chemical and Biochemical Engineering, Faculty of Engineering, Toyama University, Gofuku, Toyama 930-8555, Japan.
Taniguchi
M
M
Kitao
T
T
Morohashi
S
S
Sasakura
T
T
D000327:Adsorption; D000345:Affinity Labels / Q000737:chemistry* / Q000378:metabolism; D001671:Biochemistry / Q000379:methods; D002457:Cell Extracts / Q000737:chemistry; D002474:Cell-Free System; D011232:Chemical Precipitation; D003208:Concanavalin A / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D006358:Hot Temperature; D008320:Maltose / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012995:Solubility; D000520:alpha-Glucosidases / Q000302:isolation & purification / Q000378:metabolism
D000345:Affinity Labels; D002457:Cell Extracts; D011108:Polymers; D003208:Concanavalin A; D008320:Maltose; D000520:alpha-Glucosidases