Multistep purification of an antifreeze protein from Ammopiptanthus mongolicus by chromatographic and electrophoretic methods.
Antifreeze proteins (AFPs) are known as thermal hysteresis proteins, which can depress the freezing points of the solution by noncolligative effects, but do not affect the melting points. Although some AFPs have been found in some plants, the identity of most proteins remains unclear, owing to insufficient quantity and quality to characterize them. In this report, we describe the purification of an AFP from the winter leaves of Ammopiptanthus mongolicus using a combination of column chromatography and gel electrophoresis. After homogenization in ascorbate-acid-containing Tris buffers (pH 7.4) the soluble proteins are captured by (diethylamino)ethyl-cellulose 52 material. An elution with 0.1-0.3M KCl leads to a crude active fraction. The crude fraction is further purified on a Superdex 75 prep-grade column and finally a Poros 20HP2 column. A complex, consisting of two proteins with relative molecular masses of 34,700 and 37,100, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, is obtained by this protein purification protocol. The recovery of two proteins from the gel is carried out by electrophoresis. The purified protein, with a molecular mass of 37,100, shows thermal hysteresis activity (THA) and can modify the normal growth of ice crystals. The THA of this purified antifreeze protein is 0.24 degrees C at the concentration of 5 mg/mL.
Wang
Weixiang
W
College of Chemical Engineering, Dalian University of Technology, Dalian 116024, China. wxwang0004@sina.com.in
Wei
Lingbo
L
Wang
Guanlin
G
D021301:Antifreeze Proteins / Q000302:isolation & purification*; D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007887:Fabaceae / Q000737:chemistry*
D021301:Antifreeze Proteins