Development of supported plasma membrane platforms from giant plasma membrane vesicles to study membrane protein functions

膜蛋白是現今藥物發展中最主要的藥物作用標靶，在處理及研究這些膜蛋白的構造及功能時，保持其原本的結構及活性是難以達成的瓶頸。最主要的原因源自於膜蛋白脆弱的雙極性構造，處理過程中要避免原本位於細胞膜中之穿膜疏水構造受到水溶液環境的破壞，以免造成蛋白質構造之損毀及失活。在本研究中，我們直接從海拉細胞 (Hela cell) 發泡取出巨大細胞膜囊泡 (GPMV) 作為膜蛋白的來源，並製成支撐式脂雙層膜平台 (SLB)，如此便能將膜蛋白在原始的脂雙層膜環境中被研究，以降低蛋白質失活的可能性。我們利用螢光漂白後恢復技術 (FRAP) 觀測到這些支撐式細胞膜具有流動性，並更進一步將支撐式細胞膜建構在聚合物墊層 (polymer cushion) 上，以減少蛋白質跟基材的摩擦力，而結果的確顯示在聚乙烯馬來酸酐 (PEMA) 及聚多巴胺 (Polydopamine)上的支撐式細胞膜比在玻璃基材上的支撐式細胞膜具有更好的流動性。此外，由於在細胞雙層膜上的膜蛋白具有不對稱性，在研究我們感興趣的膜蛋白時，了解及控制蛋白質的方向性，是件重要的事情。我們利用 ADAM17 抗體的檢測結果得知，當巨大細胞膜囊泡自發性地破在基材上時，原細胞膜的內層膜會面向外部水溶液的環境。我們更發展出膜翻印的技術，以另一種與細胞膜親和力更高的基材，將原先形成的支撐式細胞膜翻面，如此一來，原本面向基材的外層細胞膜就能被暴露至外部水溶液環境中，形成外層膜向外的支撐式細胞膜平台。有了這樣的翻模技術，我們就能利用支撐式細胞膜平台對感興趣的蛋白質進行雙向的研究。Processing and handling cell membrane proteins while maintaining their intact structural information remains one of the biggest bottlenecks to characterize and understand their structure-function behavior, even though membrane proteins are the major targets for therapeutic development. Most of the problem stems from the requirement of protecting the delicate membrane-embedded hydrophobic core from water during processing to prevent denaturation and loss of function. Here, we obtained giant plasma membrane vesicles (GPMVs) directly from Hela cells and used them to form supported lipid bilayer platforms, so that the membrane proteins can be processed in their native lipid bilayer environment. The data from fluorescence recovery after photobleaching technique show that the species in the supported GPMV membrane platform have fluidity. GPMVs deposited on polymer cushions, PEMA and polydopamine, were shown to have better fluidity than those deposited on bare glass coverslips. More importantly, since the two lipid leaflets and the membrane proteins across the cell membrane are asymmetric, controlling which side of the cell membrane faces the aqueous environment and which one faces the solid support is important for us to study the interested protein function. The anti-ADAM17 antibody experiment shows that the inner leaflet faced the aqueous environment when the GPMVs spontaneously broke on the solid surface. We developed a blotting method to transfer the formed supported GPMV membrane to another suitable support to make an outer-leaflet-facing out supported GPMV membrane. With the blotting method, we might be able to use supported GPMV membranes to study membrane proteins from either side of the cell plasma membrane