Development of surface-based assays for transmembrane proteins: selective immobilization of functional CCR5, a G protein-coupled receptor.
Development of surface-based assays for transmembrane proteins: selective immobilization of functional CCR5, a G protein-coupled receptor.
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开发基于表面的跨膜蛋白检测：选择性固定功能性 CCR5（一种 G 蛋白偶联受体）。
DOI：
10.1016/j.ab.2005.10.025
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Vanderah,DavidJ
中科院分区：
文献类型：
--
作者：
Silin,VitaliiI;Karlik,EvanA;Ridge,KevinD;Vanderah,DavidJ
A general method to develop surface-based assays for transmembrane (TM) receptor function(s) without the need to isolate, purify, and reconstitute the proteins is presented. Based on the formation of an active surface that selectively immobilizes membrane vesicles, the method is illustrated using the chemokine receptor CCR5, a member of the largest family of cell surface eukaryotic TM proteins, the G protein-coupled receptors (GPCRs). The method begins with a protein-resistant surface containing a low percentage (1–5%) of surface-bound biotin on gold as the initial template. Surface plasmon resonance (SPR) data show specific immobilization of functional CCR5 after the initial template is activated by immobilization of rho 1D4 antibody, an anti-rhodopsin monoclonal antibody specific for the carboxyl terminal nine amino acids on bovine rhodopsin that had been engineered into the carboxyl terminus of CCR5, and exposure to vesicles obtained from mammalian cells transfected with a synthetic human CCR5 gene. Activation of the initial template is effected by sequential immobilization of avidin, which binds to the biotin in the initial template, a biotinylated goat anti-mouse immunoglobulin G (Bt-IgG), which binds to the avidin binding sites distal to the surface and the Fcportion of the rho 1D4 antibody through its Fabregion(s) and finally rho 1D4. This approach establishes a broad outline for the development and application of various assays for CCR5 functions. SPR data also showed that vesicle immobilization could be achieved through an integrin–integrin antibody interaction after activation of the initial template with a goat anti-human integrin β1 antibody. These results suggest that the generic nature of the initial platform and flexibility of the subsequent surface activation for specific immobilization of membrane vesicles can be applied to the development of assays for other GPCRs or TM receptors for which antibodies are available or can be engineered to contain a particular antibody epitope.
