Villaseñor Citlayi G, Karagiaridi Alexandra, Dimitrova Valentina S, Buyco Delfin G, Candal Isabella, Smirnova Anastasia, Pinkett Heather W, Kamat Neha P
Department of Biomedical Engineering, Northwestern University, Evanston, Illinois; Center of Synthetic Biology, Northwestern University, Evanston, Illinois.
Department of Molecular Biosciences, Northwestern University, Evanston, Illinois.
Biophys Rep (N Y). 2025 Jul 25;5(3):100224. doi: 10.1016/j.bpr.2025.100224.
Assessing protein insertion and association with membranes is often a critical step that follows protein synthesis for both fundamental studies on protein folding and structure as well as translational applications that harness proteins for their activity. Traditionally, membrane protein association with membranes involves ultracentrifugation, which can be time-consuming and inaccessible in low-resource scientific environments. In this study, we develop an accessible method to purify vesicle-integrated cell-free expressed proteins from unincorporated protein or lysed membranes. We use a table-top microcentrifuge, capable of reaching speeds up to 21,130 × g, and a sucrose gradient to effectively separate the bulk of the cell-free expression components from proteoliposomes. We validate our approach can be used to measure membrane association of a variety of proteins, such as peripheral and transmembrane proteins as well as lipid-specific proteins, and that our method can be extended to membrane proteins derived from cellular membranes. Our approach provides a more accessible, cost-effective, and low-volume alternative for isolating proteoliposomes from misfolded and unassociated membrane proteins that should be applicable for fundamental biophysical studies and applications involving cell-free expressed membrane proteins.
评估蛋白质插入以及与膜的结合情况，对于蛋白质折叠和结构的基础研究以及利用蛋白质活性的转化应用而言，通常是蛋白质合成后的关键步骤。传统上，膜蛋白与膜的结合涉及超速离心，这在资源匮乏的科研环境中可能既耗时又难以实现。在本研究中，我们开发了一种简便的方法，用于从未掺入的蛋白质或裂解的膜中纯化整合有囊泡的无细胞表达蛋白。我们使用一台最高转速可达21,130×g的台式微量离心机和蔗糖梯度，有效地从蛋白脂质体中分离出大部分无细胞表达成分。我们验证了我们的方法可用于测量多种蛋白质的膜结合情况，如外周蛋白和跨膜蛋白以及脂质特异性蛋白，并且我们的方法可以扩展到源自细胞膜的膜蛋白。我们的方法为从错误折叠和未结合的膜蛋白中分离蛋白脂质体提供了一种更简便、经济高效且低体积的替代方法，该方法应适用于基础生物物理研究以及涉及无细胞表达膜蛋白的应用。
