PURIFICATION AND CHARACTERIZATION OF BIOTIN-BINDING PROTEIN-II FROM CHICKEN OOCYTES
PURIFICATION AND CHARACTERIZATION OF BIOTIN-BINDING PROTEIN-II FROM CHICKEN OOCYTES
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DOI：
10.1042/bj2560797
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发表时间：
1988-12-15
影响因子：
4.1
通讯作者：
WHITE, HB
中科院分区：
文献类型：
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作者：
BUSH, L;MCGAHAN, TJ;WHITE, HB
BBP-II 是鸡卵母细胞中的主要生物素结合蛋白，经 12,000 倍纯化，产率 22%。纯化程序包括卵黄脂质的丁醇提取、水溶性蛋白质的磷酸纤维素色谱、pH 7.4的DEAE-纤维素色谱和羟基磷灰石柱色谱。通过在pH 6.0下使用第二DEAE-纤维素柱色谱法获得最终纯化。在可检测 1% 杂质的条件下，BBP-II 在聚丙烯酰胺凝胶电泳和 SDS/聚丙烯酰胺凝胶电泳上都是均质的。 SDS/聚丙烯酰胺凝胶电泳测定的亚基Mr为18,200（四聚体为72,600），与氨基酸分析计算出的Mr值17,300（69,100）相当。当针对该蛋白质的兔抗血清针对粗制鸡蛋黄样品时，形成单条沉淀素线。通过此程序纯化的 BBP-II 缺乏碳水化合物和磷酸盐，冷冻时无限期稳定，并且在室温下相当稳定。 N端氨基酸序列在前23个残基的三个位置上显示出多态性，与亲和素的N端22个残基的一致性约为45%。通过免疫扩散和酶联免疫吸附测定判断，BBP-II 抗血清与 BBP-I 以及各种鸟类和鳄鱼蛋黄中的类似蛋白质发生交叉反应。通过这两种方法均未观察到与鸡蛋清发生交叉反应。
BBP-II, the major biotin-binding protein from chicken oocytes, was purified 12,000-fold with a 22% yield. The purification procedure includes butan-1-ol extraction of yolk lipids, phosphocellulose chromatography of the water-soluble proteins, DEAE-cellulose chromatography at pH 7.4 and hydroxyapatite column chromatography. Final purification was obtained by using a second DEAE-cellulose column chromatography at pH 6.0. BBP-II was homogeneous on both polyacrylamide-gel electrophoresis and SDS/polyacrylamide-gel electrophoresis under conditions that would detect a 1% impurity. The subunit Mr determined from SDS/polyacrylamide-gel electrophoresis was 18,200 (72,600 for tetramer), which compares favourably with an Mr value of 17,300 (69,100) calculated from the amino acid analysis. A single precipitin line formed when rabbit antiserum to the protein was directed against a crude chicken egg-yolk sample. BBP-II purified by this procedure lacked carbohydrate and phosphate, was stable indefinitely when frozen, and was quite stable at room temperature. The N-terminal amino acid sequence showed polymorphism at three positions in the first 23 residues and was about 45% identical with the N-terminal 22 residues of avidin. Antiserum to BBP-II cross-reacted with BBP-I and similar proteins in the yolk of eggs from various birds and alligator as judged by immunodiffusion and enzyme-linked immunosorbent assays. No cross-reaction was observed with chicken egg-white by either of these methods.