Functional reconstitution of the HIV receptors CCR5 and CD4 in liposomes
Functional reconstitution of the HIV receptors CCR5 and CD4 in liposomes
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DOI：
10.1046/j.1432-1033.2002.03213.x
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发表时间：
2002-11-01
期刊：
影响因子：
--
通讯作者：
Poquet, Y
中科院分区：
文献类型：
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作者：
Devesa, F;Chams, V;Poquet, Y
膜蛋白的重构允许它们在可以随意操纵的膜环境中进行研究。由于膜蛋白具有不同的生物物理性质，因此迄今为止已经在特设基础上开发了针对单个蛋白的重构方法。我们开发了CCR 5的插入后重建方法，CCR 5是一种G蛋白偶联受体，具有7个跨膜α螺旋和小的外域和内域。在哺乳动物细胞中表达带His（6）标签的CCR 5，使用去污剂N-十二烷基-β-D-麦芽糖苷（DDM）纯化，并重构到用DDM饱和的预先形成的脂质体膜中，用疏水聚苯乙烯珠去除去污剂。然后，我们试图将CD 4，一种具有单一跨膜螺旋和大亲水性胞外域的蛋白质与CCR 5一起掺入脂质体膜中。令人惊讶的是，该蛋白质的重构也通过该方法实现。发现两种蛋白质一起存在于单独的脂质体中。重组的CCR 5被几种单克隆抗体识别，识别其天然配体，CD 4结合可溶性形式的gp 120，gp 120是HIV融合蛋白的一个亚基，使用CD 4作为受体。此外，表达HIV完整融合蛋白的细胞与脂质体结合，表明蛋白质是完整的，并且大多数蛋白质都是正面向外的。因此，两个广泛不同的蛋白质的功能共重构可以通过这种方法来实现，这表明它可能是有用的其他蛋白质。
Reconstitution of membrane proteins allows their study in a membrane environment that can be manipulated at will. Because membrane proteins have diverse biophysical properties, reconstitution methods have so far been developed for individual proteins on an ad hoc basis. We developed a postinsertion reconstitution method for CCR5, a G protein coupled receptor, with seven transmembrane alpha helices and small ecto- and endodomains. A His(6)-tagged version of CCR5 was expressed in mammalian cells, purified using the detergent N-dodecyl-beta-D-maltoside (DDM) and reconstituted into preformed liposomal membranes saturated with DDM, removing the detergent with hydrophobic polystyrene beads. We then attempted to incorporate CD4, a protein with a single transmembrane helix and a large hydrophilic ectodomain into liposomal membranes, together with CCR5. Surprisingly, reconstitution of this protein was also achieved by the method. Both proteins were found to be present together in individual liposomes. The reconstituted CCR5 was recognized by several monoclonal antibodies, recognized its natural ligand, and CD4 bound a soluble form of gp120, a subunit of the HIV fusion protein that uses CD4 as a receptor. Moreover, cells expressing the entire fusion protein of HIV bound to the liposomes, indicating that the proteins were intact and that most of them were oriented right side out. Thus, functional coreconstitution of two widely different proteins can be achieved by this method, suggesting that it might be useful for other proteins.