Smith Sinead Marian
Institute of Molecular Medicine and Immunology Research Centre, Trinity College Dublin, Dublin, Ireland.
Methods Mol Biol. 2011;681:485-96. doi: 10.1007/978-1-60761-913-0_29.
Although membrane proteins account for 20-30% of the coding regions of all sequenced genomes and play crucial roles in many fundamental cell processes, there are relatively few membranes proteins with known 3D structure. This is likely due to technical challenges associated with membrane protein extraction, solubilisation, and purification. Membrane proteins are classified based on the level of interaction with membrane lipid bilayers, with peripheral membrane proteins associating non-covalently with the membrane, and integral membrane proteins associating more strongly by means of hydrophobic interactions. Generally speaking, peripheral membrane proteins can be purified by milder techniques than integral membrane proteins, whose extraction requires phospholipid bilayer disruption by detergents. Here, important criteria for strategies of membrane protein purification are addressed, with a focus on the initial stages of membrane protein solublilisation, where problems are most frequently encountered. Protocols are outlined for the successful extraction of peripheral membrane proteins, solubilisation of integral membrane proteins, and detergent removal which is important not only for retaining native protein stability and biological functions, but also for the efficiency of later purification techniques.
尽管膜蛋白占所有已测序基因组编码区的20%-30%，并在许多基本细胞过程中发挥关键作用，但具有已知三维结构的膜蛋白相对较少。这可能是由于与膜蛋白提取、溶解和纯化相关的技术挑战。膜蛋白根据与膜脂双层的相互作用水平进行分类，外周膜蛋白通过非共价方式与膜结合，而整合膜蛋白则通过疏水相互作用更紧密地结合。一般来说，外周膜蛋白可以通过比整合膜蛋白更温和的技术进行纯化，整合膜蛋白的提取需要用去污剂破坏磷脂双层。本文讨论了膜蛋白纯化策略的重要标准，重点关注膜蛋白溶解的初始阶段，这是最常遇到问题的地方。文中概述了成功提取外周膜蛋白、溶解整合膜蛋白以及去除去污剂的方案，去污剂的去除不仅对于保持天然蛋白的稳定性和生物学功能很重要，而且对于后续纯化技术的效率也很重要。
