Expression, surface immobilization, and characterization of functional recombinant cannabinoid receptor CB2.
Expression, surface immobilization, and characterization of functional recombinant cannabinoid receptor CB2.
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DOI：
10.1016/j.bbapap.2013.06.003
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发表时间：
2013-10
影响因子：
3.2
通讯作者：
Yeliseev, Alexei A.
中科院分区：
文献类型：
--
作者：
Locatelli-Hoops, Silvia C.;Gorshkova, Inna;Gawrisch, Klaus;Yeliseev, Alexei A.
人外周大麻素受体CB2是一种参与免疫应答调控的G蛋白偶联受体（GPCR），已成为药物开发的重要靶点。CB2的结构和功能研究可能受益于纯化和功能受体以控制密度固定在合适的表面上，最好是在均匀的方向上。该项目的目标是开发一种通用策略，用于制备功能性重组CB2和在固体界面上固定。在大肠杆菌中，CB2与Rho-tag（由rhodopsin的后9个氨基酸组成的肽）融合后的表达在蛋白水平、标签的可及性和受体的活性方面进行了评估。CB2的结构完整性通过与受体结合的配体来测试，该受体溶解在洗涤剂胶束中，在标签特异性单克隆1D4抗体包被树脂上捕获。通过在1D4-和Ni-NTA-树脂上的序列层析获得了高纯度和功能性的CB2，并通过表面等离子体共振（SPR）表征了其与1D4抗体的亲和力。从粗细胞提取物中纯化的受体或融合的CB2被捕获到1D4包被的CM4芯片（Biacore）上，以定量的方式以均匀的方向被SPR信号所证明。此外，通过SPR研究了固定化CB2在细胞外表面的可及性以及与新型单克隆抗体NAA-1相互作用的亲和力。综上所述，我们提出了纯化、表面固定化、配体和抗体结合研究功能性大麻素受体CB2的整体策略。
Human peripheral cannabinoid receptor CB2, a G protein-coupled receptor (GPCR) involved in regulation of immune response has become an important target for pharmaceutical drug development. Structural and functional studies on CB2 may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. The goal of this project was to develop a generic strategy for preparation of functional recombinant CB2 and immobilization at solid interfaces. Expression of CB2 as a fusion with Rho-tag (peptide composed of the last nine amino acids of rhodopsin) in E. coli was evaluated in terms of protein levels, accessibility of the tag, and activity of the receptor. The structural integrity of CB2 was tested by ligand binding to the receptor solubilized in detergent micelles, captured on tag-specific monoclonal 1D4 antibody-coated resin. Highly pure and functional CB2 was obtained by sequential chromatography on a 1D4- and Ni-NTA- resin and its affinity to the 1D4 antibody characterized by surface plasmon resonance (SPR). Either the purified receptor or fusion CB2 from the crude cell extract was captured onto a 1D4 -coated CM4 chip (Biacore) in a quantitative fashion at uniform orientation as demonstrated by the SPR signal. Furthermore, the accessibility of the extracellular surface of immobilized CB2 and the affinity of interaction with a novel monoclonal antibody NAA-1 was studied by SPR. In summary, we present an integral strategy for purification, surface immobilization, ligand- and antibody binding studies of functional cannabinoid receptor CB2.
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DOI：
10.1073/pnas.190274097
发表时间：
2000-10-10
影响因子：
11.1
作者：
Hoffman, TL;Canziani, G;Doms, RW
通讯作者：
Doms, RW
影响因子：
1.6
作者：
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影响因子：
9.9
作者：
STENBERG, E;PERSSON, B;URBANICZKY, C
通讯作者：
URBANICZKY, C
影响因子：
2.9
作者：
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通讯作者：
Winzor, DJ
DOI：
10.1073/pnas.84.24.8874
发表时间：
1987-12-01
影响因子：
11.1
作者：
OPRIAN, DD;MOLDAY, RS;KHORANA, HG
通讯作者：
KHORANA, HG