Purification of Membrane Proteins

Abstract

In contrast to soluble proteins, the isolation of a peripheral or integral membrane protein requires extraction of the biological membrane containing the protein of interest prior to purification. The most commonly used extraction procedures to isolate membrane proteins are described in Chapter 29. Following initial solubilization in high concentrations of detergent (1), intrinsic proteins can then be purified to homogeneity using a variety of biochemical techniques in the presence of relatively low concentrations of detergent. Solubilized membrane proteins from diverse sources and with different properties have successfully been purified by a combination of standard techniques such as density gradient centrifugation, ion-exchange chromatography, gel filtration, lectin chromatography, and different forms of the affinity chromatography method. Usually, separation techniques based on biological differences such as affinity chromatography using specific ligands, probes, or antibodies highly specific for a membrane protein result in a higher yield and purity of the isolated protein than chromatographical methods based on physical differences, such as size and charge of the membrane protein (2,3). An especially powerful separation technique for the initial purification of membrane-associated glycoproteins is lectin affinity chromotography. As described in more detail in Chapter 18, this method explores the highly specific interaction between N- and O-linked oligosaccharide chains on glycosylated proteins with immobilized lectins, which usually results in a remarkable enrichment of integral glycoproteins (4).