Analysis of membrane protein complex using baculoviral display

使用杆状病毒展示分析膜蛋白复合物

• 批准号: 16310147
• 负责人: HAMAKUBO Takao
• 金额: $ 10.05万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16310147/
• 关键词: baculovirus; membrane protein; GPCR; γ-secretase; lipid raft; nicastrin; peptide transporter; FRET; 核内受容体; 磁性ビーズ; プロテオミクス; 転写複合体; HNF4α; ラフト; スタチン; アルツハイマー

Membrane proteins such as peptide transporter G-protein coupled receptors (BLT1, CCR2, CCR5, Edg1, AT1a, AT2, D1, D5) and their complex (reoeptor-Gs-adenylyl cyclase) or active γ-secretase complex (presenillin-APH1-nicastrin-PEN2) have been successfully recovered in baculovirus fraction. The expressed membrane proteins were considered to be active on the baculovirus membrane. Interestingly, γ -secretase acitivity was recovered only when all 4 components were expressed on the baculovirus. Using these membrane protein expressed baculovirus, specific monoclonal antibodies (Mabs) were generated against membrane proteins including GPCR. These Mabs are usable not only for western blotting but also FACS analysis and immunohistochemistry. Some of them have shown the neutralizing activity against ligand-receptor mediated cAMP production or the antibody dependent cell cytotoxic activities. Also we showed the importance of lipid micro- environment of membrane and geranyl-geranylation for γ-secretase acitivity in generating β-amyloid from its precursor. One molecule FRET(fluorescence resonance energy transfer) was succefully constructed using RGS (regulator of G- protein signalling) and Gi which recognized G- protein activation by alminium fluoride.These results indicate that the baculovirus display system of membrane proteins or their complex is useful not only for high throughput screening assay system but also for making active Mabs for therapeutic use.

已成功地从杆状病毒组分中回收了膜蛋白，如肽转运蛋白G蛋白偶联受体（BLT 1、CCR 2、CCR 5、Edg 1、AT 1a、AT 2、D1、D5）及其复合物（受体-Gs-腺苷酸环化酶）或活性γ-分泌酶复合物（早老素-APH 1-nicastrin-PEN 2）。表达的膜蛋白被认为在杆状病毒膜上具有活性。有趣的是，只有当所有4种组分在杆状病毒上表达时，γ -分泌酶活性才恢复。使用这些表达膜蛋白的杆状病毒，产生针对膜蛋白（包括GPCR）的特异性单克隆抗体（Mab）。这些单克隆抗体不仅可用于蛋白质印迹，而且可用于流式细胞仪分析和免疫组织化学。它们中的一些已经显示出对配体-受体介导的cAMP产生的中和活性或抗体依赖性细胞毒性活性。我们还发现膜的脂质微环境和香叶基香叶基化对γ-分泌酶活性在从其前体生成β-淀粉样蛋白中的重要性。利用G蛋白信号调节子（regulator of G- protein signaling，RGS）和识别氟化铝激活的G蛋白的Gi，成功地构建了一个荧光共振能量转移（fluorescence resonance energy transfer，FRET）分子，这些结果表明，杆状病毒膜蛋白或膜蛋白复合物的展示系统不仅可用于高通量筛选检测系统，而且可用于制备具有治疗活性的单克隆抗体。

项目成果

Identification of soluble NH2-terminal fragment of glypican-3 as a serological marker for early-stage hepatocellular carcinoma

• DOI: 10.1158/0008-5472.can-03-2191
• 发表时间: 2004-04-01
• 期刊: CANCER RESEARCH
• 影响因子: 11.2
• 作者: Hippo, Y;Watanabe, K;Aburatani, H
• 通讯作者: Aburatani, H

Dysregulated expression of P1 and P2 promoter-driven hepatocyte nuclear factor-4alpha in the pathogenesis of human cancer.

P1 和 P2 启动子驱动的肝细胞核因子 4α 在人类癌症发病机制中的表达失调。

• 发表时间: 2006
• 期刊: J Pathol. 208(5)
• 作者: Tanaka T;Jiang S;Hotta H;Takano K;Iwanari H;Sumi K;Daigo K;Ohashi R;Sugai M;Ikegame C;Umezu H;Hirayama Y;Midorikawa Y;Hippo Y;Watanabe A;Uchiyama Y;Hasegawa G;Reid P;Aburatani H;Hamakubo T;Sakai J;Naito M;Kodama T.
• 通讯作者: Kodama T.