O'Riordan C R, Erickson A, Bear C, Li C, Manavalan P, Wang K X, Marshall J, Scheule R K, McPherson J M, Cheng S H
Genzyme Corporation, Framingham, Massachusetts 01701-9322, USA.
J Biol Chem. 1995 Jul 14;270(28):17033-43. doi: 10.1074/jbc.270.28.17033.
We have developed procedures to purify highly functional recombinant cystic fibrosis transmembrane conductance regulator (CFTR) from Chinese hamster ovary (CHO) cells to high homogeneity. Purification of CHO-CFTR was achieved using a combination of alkali stripping, alpha-lysophosphatidylcholine extraction, DEAE ion-exchange, and immunoaffinity chromatography. Insect CFTR from Sf9 cells was purified using a modification of the method of Bear et al. (Bear, C. E., Li, C., Kartner, N., Bridges, R. J., Jensen, T. J., Ramjeesingh, M. and Riordan, J. R. (1992) Cell 68, 809-818), which included extraction with sodium dodecyl sulfate, hydroxyapatite, and gel filtration chromatography. Characterization of the properties of purified CFTR from both cell sources using a variety of electrophysiological and biochemical assays indicated that they were very similar. Both the purified CHO-CFTR and Sf9-CFTR when reconstituted into planar lipid bilayers exhibited a low pS, chloride-selective ion channel activity that was protein kinase A- and ATP-dependent. Both the purified CHO-CFTR and Sf9-CFTR were able to interact specifically with the nucleotide photoanalogue 8-N3-[alpha-32P]ATP with half-maximal binding at 25 and 50 microM, respectively. These values compare well with those reported for 8-N3-[alpha-32P]ATP binding to CFTR in its native membrane form. Thus CFTR from either insect or CHO cells can be purified to high homogeneity with retention of many of the biochemical and electrophysiological characteristics of the protein associated in its native plasma membrane form. The availability of these reagents will facilitate further investigation and study of the structure and function of CFTR and its interactions with cellular proteins.
我们已开发出从中国仓鼠卵巢（CHO）细胞中纯化高功能重组囊性纤维化跨膜传导调节因子（CFTR）至高纯度的方法。使用碱剥离、α-溶血磷脂酰胆碱提取、DEAE离子交换和免疫亲和色谱相结合的方法实现了CHO-CFTR的纯化。来自Sf9细胞的昆虫CFTR使用Bear等人（Bear, C. E., Li, C., Kartner, N., Bridges, R. J., Jensen, T. J., Ramjeesingh, M. and Riordan, J. R. (1992) Cell 68, 809 - 818）方法的改进版本进行纯化，该方法包括用十二烷基硫酸钠提取、羟基磷灰石和凝胶过滤色谱。使用多种电生理和生化测定对来自两种细胞来源的纯化CFTR的特性进行表征表明它们非常相似。纯化的CHO-CFTR和Sf9-CFTR在重构到平面脂质双分子层中时均表现出低皮安、氯离子选择性离子通道活性，该活性依赖于蛋白激酶A和ATP。纯化的CHO-CFTR和Sf9-CFTR都能够与核苷酸光类似物8-N3-[α-32P]ATP特异性相互作用，其半最大结合浓度分别为25和50微摩尔。这些值与报道的8-N3-[α-32P]ATP与天然膜形式的CFTR结合的值相当。因此，来自昆虫或CHO细胞的CFTR都可以纯化至高纯度，并保留其在天然质膜形式中相关蛋白质的许多生化和电生理特性。这些试剂的可得性将有助于进一步研究CFTR的结构和功能及其与细胞蛋白的相互作用。
