Hofmann K, Wood S W, Brinton C C, Montibeller J A, Finn F M
Proc Natl Acad Sci U S A. 1980 Aug;77(8):4666-8. doi: 10.1073/pnas.77.8.4666.
A method is described for the retrieval of streptavidin from the culture broth of Streptomyces avidinii. The key step in this procedure is the adsorption of streptavidin from culture concentrates to an affinity column in which iminobiotin is attached to AH-Sepharose 4B. This column binds streptavbidin at pH 11 and releases the protein at pH 4. The recovery of streptavidin is practically quantitative. The pH dependence of the iminobiotin-avidin affinity, discovered by Green [Green, N. M. (1966) Biochem. J. 101, 774-779], has thus found practical application. The streptavidin bound 4.07 +/- 0.02 mol of [14C]biotin per mol and was essentially homogeneous as judged by disc and slab gel electrophoresis. Streptavidin was extensively succinoylated without loss of biotin-binding capacity. The observations that 125I-labeled streptavidin and 125I-labeled succinoylstreptavidin are retained by iminobiotin-AH-Sepharose 4B columns at pH 7.5 and are eluted at pH 4.0 provides a convenient purification method for these iodinated proteins. The technique employed for the retrieval of streptavidin is generally applicable to the isolation of iminobiotinylated molecules.
描述了一种从抗生物素链霉菌的培养液中提取链霉亲和素的方法。该方法的关键步骤是将链霉亲和素从培养浓缩物中吸附到亲和柱上，该亲和柱上的亚氨基生物素连接到AH-琼脂糖4B上。此柱在pH 11时结合链霉亲和素，在pH 4时释放蛋白质。链霉亲和素的回收率几乎是定量的。格林[格林，N.M.（1966年）《生物化学杂志》101卷，774 - 779页]发现的亚氨基生物素 - 抗生物素蛋白亲和力的pH依赖性因此得到了实际应用。每摩尔链霉亲和素结合4.07±0.02摩尔的[¹⁴C]生物素，通过圆盘和平板凝胶电泳判断其基本均一。链霉亲和素被广泛琥珀酰化而不丧失生物素结合能力。观察到¹²⁵I标记的链霉亲和素和¹²⁵I标记的琥珀酰化链霉亲和素在pH 7.5时被亚氨基生物素 - AH - 琼脂糖4B柱保留，在pH 4.0时被洗脱，这为这些碘化蛋白提供了一种便捷的纯化方法。用于提取链霉亲和素的技术通常适用于亚氨基生物素化分子的分离。
