Published:Journal of Chromatographic Science,
ISSN 0021-9665Volume
41, Number 9, October 2003, pp. 489-493
Multistep Purification of an Antifreeze Protein from Ammopiptanthus
mongolicus by Chromatographic and Electrophoretic Methods
Weixiang Wang[1],[2], Lingbo Wei[2], and Guanlin Wang[1]
[1]College of Chemical Engineering, Dalian University of Technology, Dalian
116024, China and
[2]Institute of Genetic and Developmental Biology, Chinese
Academy
of Sciences, Beijing 100080, China
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°C at the concentration of 5 mg/mL.
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