Insertion of membrane proteins into discoidal membranes using a cell-free protein expression approach
Insertion of membrane proteins into discoidal membranes using a cell-free protein expression approach
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DOI：
10.1021/pr800265f
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发表时间：
2008-08-01
影响因子：
4.4
通讯作者：
Kudlicki, Wieslaw
中科院分区：
文献类型：
--
作者：
Katzen, Federico;Fletcher, Julia E.;Kudlicki, Wieslaw
我们报告了一种无细胞方法，用于表达整合膜蛋白并将其插入由盘状载脂蛋白-脂质双层组成的水溶性颗粒中。将蛋白质插入颗粒中，避免了将产品提取并重构为膜囊泡的需要。此外，膜支持物的平面性质使得蛋白质可以从脂双层的两侧自由地进入。通过载脂蛋白成分或载体蛋白成功地纯化了复合物。该方法显着增强了具有不同功能作用和拓扑结构的多种膜蛋白的溶解度。对模型膜蛋白子集的分析测定表明蛋白质正确折叠并具有活性。该方法提供了一个平台，可以对各种传统上棘手的药物靶标进行高通量结构和功能表征。
We report a cell-free approach for expressing and inserting integral membrane proteins into water-soluble particles composed of discoidal apolipoprotein-lipid bilayers. Proteins are inserted into the particles, circumventing the need of extracting and reconstituting the product into membrane vesicles. Moreover, the planar nature of the membrane support makes the protein freely accessible from both sides of the lipid bilayer. Complexes are successfully purified by means of the apoplipoprotein component or by the carrier protein. The method significantly enhances the solubility of a variety of membrane proteins with different functional roles and topologies. Analytical assays for a subset of model membrane proteins indicate that proteins are correctly folded and active. The approach provides a platform amenable to high-throughput structural and functional characterization of a variety of traditionally intractable drug targets.