Huang P, Liu Q, Scarborough G A
Department of Pharmacology, University of North Carolina, Chapel Hill 27599, USA.
Anal Biochem. 1998 May 15;259(1):89-97. doi: 10.1006/abio.1998.2633.
Similar to the recombinant cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Sf9 insect cells, underglycosylated CFTR expressed in yeast is not effectively solubilized by a variety of commonly used detergents, requiring instead harsh alkali and SDS treatments, which would denature most proteins. Moreover, solubilized CFTR has a strong tendency to aggregate and form high-molecular-weight aggregates during subsequent purification. We report here that the mild detergent, lysophosphatidylglycerol (LPG), is a very effective detergent for solubilizing the CFTR expressed in both yeast and Sf9 insect cells. LPG solubilizes nearly 100% of the CFTR in yeast in the absence of NaCl and none in the presence of 1 M NaCl. It is also very potent in preventing aggregation of the CFTR during subsequent purification. Exploiting these characteristics, a rapid simple procedure for the purification of functional recombinant CFTR expressed in yeast has been developed. It includes selective CFTR solubilization in the presence and the absence of NaCl followed by nickel-chelate chromatography of His-tagged CFTR. The CFTR produced by this procedure is about 70% pure. Purified CFTR molecules were reconstituted into liposomes and then fused to planar lipid bilayers for single-channel recording. The reconstituted CFTR exhibits regulatory chloride channel activities with a slope conductance of 7.1 pS and a reversal potential of -32 mV. The effectiveness and simplicity of this new purification procedure for the CFTR should greatly facilitate a variety of biochemical and biophysical studies of this important protein. Furthermore, the potency of LPG in solubilizing the notoriously intractable underglycosylated CFTR suggest that this detergent may be useful for solubilizing the CFTR from other sources and for other difficult membrane proteins as well.
与在Sf9昆虫细胞中表达的重组囊性纤维化跨膜传导调节因子（CFTR）相似，在酵母中表达的低糖基化CFTR不能被多种常用去污剂有效溶解，而是需要苛刻的碱和SDS处理，而这会使大多数蛋白质变性。此外，在随后的纯化过程中，溶解的CFTR有强烈的聚集倾向并形成高分子量聚集体。我们在此报告，温和去污剂溶血磷脂酰甘油（LPG）是溶解在酵母和Sf9昆虫细胞中表达的CFTR的非常有效的去污剂。在不存在NaCl的情况下，LPG可溶解酵母中近100%的CFTR，而在存在1 M NaCl的情况下则不能溶解任何CFTR。它在防止CFTR在随后的纯化过程中聚集方面也非常有效。利用这些特性，开发了一种快速简单的方法来纯化在酵母中表达的功能性重组CFTR。它包括在存在和不存在NaCl的情况下选择性溶解CFTR，然后对带有His标签的CFTR进行镍螯合层析。通过该方法产生的CFTR纯度约为70%。纯化的CFTR分子被重组到脂质体中，然后与平面脂质双层融合以进行单通道记录。重组的CFTR表现出调节性氯离子通道活性，斜率电导为7.1 pS，反转电位为-32 mV。这种用于CFTR的新纯化方法的有效性和简单性应极大地促进对这种重要蛋白质的各种生化和生物物理研究。此外，LPG溶解 notoriously intractable 的低糖基化CFTR的能力表明，这种去污剂可能也有助于从其他来源溶解CFTR以及其他难处理的膜蛋白。
