Strategies for the Purification of Membrane Proteins英文文献.pdfVIP

Chapter 29 Strategies for the Purification of Membrane Proteins Sinead Marian Smith Abstract 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 prateins 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 func- tions, but also for the efficiency of later purification techniques. Key words: Peripheral membrane protein, Integral membrane protein, Detergent, Protein purifica- tion, Protein solubilisation 1. Introduction Membrane proteins are associated with the membrane of a cell or particular organelle and are generally more problematic to purify than water-soluble proteins. Membrane proteins represent