Mirzabekov T, Kontos H, Farzan M, Marasco W, Sodroski J
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115, USA.
Nat Biotechnol. 2000 Jun;18(6):649-54. doi: 10.1038/76501.
Seven-transmembrane segment, G protein-coupled receptors play central roles in a wide range of biological processes, but their characterization has been hindered by the difficulty of obtaining homogeneous preparations of native protein. We have created paramagnetic proteoliposomes containing pure and oriented CCR5, a seven-transmembrane segment protein that serves as the principal coreceptor for human immunodeficiency virus (HIV-1). The CCR5 proteoliposomes bind the HIV-1 gp120 envelope glycoprotein and conformation-dependent antibodies against CCR5. The binding of gp120 was enhanced by a soluble form of the other HIV-1 receptor, CD4, but did not require additional cellular proteins. Paramagnetic proteoliposomes are uniform in size, stable in a broad range of salt concentrations and pH, and can be used in FACS and competition assays typically applied to cells. Integral membrane proteins can be inserted in either orientation into the liposomal membrane. The magnetic properties of these proteoliposomes facilitate rapid buffer exchange useful in multiple applications. As an example, the CCR5-proteoliposomes were used to select CCR5-specific antibodies from a recombinant phage display library. Thus, paramagnetic proteoliposomes should be useful tools in the analysis of membrane protein interactions with extracellular and intracellular ligands, particularly in establishing screens for inhibitors.
七跨膜段G蛋白偶联受体在广泛的生物过程中发挥核心作用，但其特性研究因难以获得天然蛋白的均一制剂而受阻。我们制备了含有纯化且定向的CCR5的顺磁蛋白脂质体，CCR5是一种七跨膜段蛋白，作为人类免疫缺陷病毒（HIV-1）的主要共受体。CCR5蛋白脂质体可结合HIV-1 gp120包膜糖蛋白以及针对CCR5的构象依赖性抗体。gp120的结合可被另一种HIV-1受体CD4的可溶性形式增强，但不需要其他细胞蛋白。顺磁蛋白脂质体大小均匀，在广泛的盐浓度和pH范围内稳定，可用于通常应用于细胞的流式细胞术（FACS）和竞争分析。整合膜蛋白可以以任何一种方向插入脂质体膜中。这些蛋白脂质体的磁性特性有助于在多种应用中进行快速缓冲液交换。例如，CCR5蛋白脂质体被用于从重组噬菌体展示文库中筛选CCR5特异性抗体。因此，顺磁蛋白脂质体应是分析膜蛋白与细胞外和细胞内配体相互作用的有用工具，特别是在建立抑制剂筛选方面。
