Kohanski R A, Lane M D
Ann N Y Acad Sci. 1985;447:373-85. doi: 10.1111/j.1749-6632.1985.tb18452.x.
Insulin receptor was purified in high yield from cultured 3T3-L1 mouse adipocytes using the bifunctional ligand N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin in conjunction with avidin-Sepharose CL-4B. This ligand is 100% competent as insulin and 60% competent as biotin, as measured by competitive binding assays. The procedure requires preliminary removal of biotin-containing proteins on "native" avidin-Sepharose CL-4B. This matrix shows nearly the same biotin-binding characteristics as uncoupled avidin and can be regenerated by washing with 0.02 N HCl, causing only a minor loss of nonexchangeable biotin-binding sites. Insulin receptor is isolated by formation of a complex between the bifunctional ligand and the receptor, and then adsorption to "monomeric" avidin-Sepharose via the biotin moiety. This affinity matrix binds [14C]biotin with a Kd approximately equal to 0.2 microM and has exchangeable/nonexchangeable biotin binding sites in the ratio 9:1. Displacement of homogeneous insulin receptor is achieved by the addition of biotin; the elution is time-dependent, suggesting that it is accomplished by the prevention of rebinding.
利用双功能配体NαB1-(生物素基-ε-氨基己酰基)胰岛素结合抗生物素蛋白-琼脂糖CL-4B，从培养的3T3-L1小鼠脂肪细胞中高产率地纯化胰岛素受体。通过竞争性结合试验测定，该配体作为胰岛素时活性为100%，作为生物素时活性为60%。该方法需要先用“天然”抗生物素蛋白-琼脂糖CL-4B初步去除含生物素的蛋白质。这种基质显示出与未偶联的抗生物素蛋白几乎相同的生物素结合特性，并且可以通过用0.02N HCl洗涤来再生，仅导致少量不可交换生物素结合位点的损失。通过双功能配体与受体形成复合物，然后通过生物素部分吸附到“单体”抗生物素蛋白-琼脂糖上来分离胰岛素受体。这种亲和基质以大约等于0.2μM的Kd结合[14C]生物素，并且具有可交换/不可交换生物素结合位点，其比例为9:1。通过添加生物素实现均一胰岛素受体的置换；洗脱是时间依赖性的，这表明它是通过防止重新结合来完成的。
