利用吉利丁翻膜技術翻轉支撐式細胞膜中之脂質與蛋白質面向

Abstract

在細胞的生理現象中，膜蛋白扮演極重要的角色。然而在傳統膜蛋白的研究上，常先將膜蛋白從細胞膜中取出，將之分離純化後再插入人工脂質膜進行研究，而膜蛋白雙親性的不穩定結構常造成分離純化過程中潛在的膜蛋白失活風險，且膜蛋白插入人工脂質球的面相亦在實驗中難以控制，增加研究膜蛋白的困難。因此，近期許多研究開始致力於發展將膜蛋白維持在原生膜環境中進行膜蛋白研究的方法，以此維持雙親性膜蛋白的活性與統一的膜蛋白面向。在本實驗中，我們直接從海拉細胞中（Hela cell）取得從膜發出含有原生膜蛋白的巨大細胞囊泡(GPMV)，將此巨大細胞囊泡在玻璃上鋪成支撐式細胞膜平台，並展示這種支撐式細胞膜平台具有統一的細胞膜面向。此外，為了同時對細胞膜兩側的蛋白進行雙向研究，我們發展出一吉利丁翻膜技術，來控制細胞膜在基材上的面向：我們先將巨大細胞囊泡鋪在吉利丁上形成支撐式細胞膜平台，再用重力壓印的方式，將吉利丁上的支撐式細胞膜轉印至新的基材上以翻轉細胞膜與膜蛋白的面向。我們利用海拉細胞膜中富含的一種穿膜蛋白－水通道蛋白3 (Aquaporin 3) 作為免疫染色的抗體標靶，來檢測直接鋪於基材與翻膜後基材上支撐式細胞膜的面向，藉由分別針對水通道蛋白3在細胞膜內側或細胞膜外側結構的抗體結合結果，可推知巨大細胞囊泡直接鋪在基材上的面向為原細胞膜內側朝上，而透過吉利丁翻膜技術將細胞膜轉印到基材上的面向為原細胞膜外側朝上。此外,我們透過調整吉利丁種類與環境酸鹼值來改變吉利丁表面電荷性質,以達到更好的細胞膜覆蓋率以及翻膜的效率。透過這套吉利丁翻膜技術，我們可以在實驗中控制基材上支撐式細胞膜的面向，並藉此同時研究細胞膜蛋白兩側的性質與功能。

Membrane proteins play important roles in many cellular processes. Conventional methods often extract membrane proteins from cell membranes and reconstitute them into artificial liposomes to study membrane proteins. However, amphiphilic membrane proteins may denature during the extraction process, and it is also a challenge to control the inserted orientation of membrane proteins in artificial liposomes. Methods that can retain membrane protein structure and orientation in their native cell plasma membrane are desirable for membrane protein studies. Here, we derived giant plasma membrane vesicles (GPMV) directly from Hela cells in order to retain the protein structure and orientation, and the supported cell membrane platforms formed by GPMV deposition showed consistent orientation of membrane proteins. In addition, to study membrane proteins from either cytoplasmic side or extracellular side, we constructed a gelatin blotting method to reverse the orientation of the supported cell membrane. We first deposited GPMV on gelatin to form the supported cell membrane, and then blotted it to a glass coverslip to obtain the supported cell membrane with reversed orientation. We used Aquaporin 3, an abundant native transmembrane protein in Hela cells, as a target to examine the topology of AQP3 in either directly deposited or blotted cell membranes. The immunostaining results of AQP3 showed that the intracellular side of the protein faced to the bulk solution in the directly-deposited membrane, while the extracellular side of the protein faced to the bulk solution in the blotted membrane. We further improved the supported cell membrane coverage on gelatin and the blotting efficiency by adjusting the gelatin type and pH condition, which are factors that influence the electrostatic properties of gelatin. With the developed gelatin blotting methods, we can control the orientation of lipids and membrane proteins in supported cell membranes and study membrane protein properties and functions from either the cytoplasmic side or extracellular side.