摘要
将ＡＢＡ通过一个１０ 碳原子的臂高效率地（６～８ ｍｍｏｌ／Ｌ凝胶）偶联到琼脂糖凝胶４Ｂ上，制成亲和层析柱，用以纯化蚕豆（Ｖｉｃｉａｆａｂａ Ｌ．） 叶片下表皮ＡＢＡ 结合蛋白（ＡＢＡ＿ＢＰ）。样品上柱后，通过ＮａＣｌ 盐梯度洗脱去掉大部分非特异结合的杂蛋白后，用１ ｍｍｏｌ／ＬＡＢＡ亲和洗脱竞争亲和柱中的ＡＢＡ＿ＢＰ，纯化到一种ＡＢＡ特异结合蛋白，纯化了１１２ 倍。纯化蛋白与ＡＢＡ 最大结合为５８．３３ ｎｍｏｌ／ｇ ｐｒｏｔｅｉｎ，Ｋｄ 值为２１ ｎｍｏｌ／Ｌ，亚基分子量为４４ ．２ ｋＤ，纯度约为９０ ％ 。纯化的ＡＢＡ结合蛋白具有典型的蛋白质紫外吸收光谱，在２８０ ｎｍ
Abscisic acid (ABA) was efficiently cross_linked to Sepharose 4B (6～8 mmol ABA/L gel) by an arm of 10_atom carbon chain. Solubilized ABA_BP (ABA binding protein) was allowed to bind to the gel, while unrelated proteins were removed by washing with a gradient of NaCl buffer. The ABA_BP was eluted with 1 mmol/L ABA. Since ABA at high concentration can interfere with both the binding activity assay and protein analysis, the fractions eluted with ABA were passed through a Sephadex G_25 column to remove the ABA. Fractions containing the binding activity were pooled, concentrated with ultra_filtration. The maximum binding capacity ( B MAX ) of the purified ABA_BP was 58.33 nmol/g protein, and the K d was 21 nmol/L, with an approximately 112 folds increase of purity. SDS_PAGE identification of the purified ABA_BP revealed a major protein band with a molecular weight of about 44.2 kD, and a purity of approximately 90%.
基金
国家自然科学基金
关键词
ABA
结合蛋白
亲和层析
蛋白纯化
ABA, Binding protein, Affinity chromatography, Purification of protein
