Mechanisms of phase behaviour and protein partitioning in detergent/polymer aqueous two-phase systems for purification of integral membrane proteins

Detergent/polymer aqueous two-phase systems are studied as a fast, mild and efficient general separation method for isolation of labile integral membr ane proteins. Mechanisms for phase behaviour and protein partitioning of bo th membrane-bound and hydrophilic proteins have been examined in a large nu mber of detergent/polymer aqueous two-phase systems. Non-ionic detergents s uch as the Triton series (polyoxyethylene alkyl phenols), alkyl polyoxyethy lene ethers (CmEOn), Tween series (polyoxyethylene sorbitol esters) and alk ylglucosides form aqueous two-phase systems in mixtures with hydrophilic po lymers, such as PEG or dextran, at low and moderate temperatures. Phase dia grams for these mixtures are shown and phase behaviour is discussed from a thermodynamic model. Membrane proteins, such as bacteriorhodopsin and chole sterol oxidase, were partitioned strongly to the micelle phase, while hydro philic proteins, BSA and lysozyme, were partitioned to the polymer phase. T he partitioning of membrane protein is mainly determined by non-specific hy drophobic interactions between detergent and membrane protein. An increased partitioning of membrane proteins to the micelle phase was found with an i ncreased detergent concentration difference between the phases, lower polym er molecular weight and increased micelle size. Partitioning of hydrophilic proteins is mainly related to excluded volume effects, i.e. increased phas e component size made the hydrophilic proteins partition more to the opposi te phase. Addition of ionic detergent to the system changed the partitionin g of membrane proteins slightly, but had a strong effect on hydrophilic pro teins, and can be used for enhanced separation between hydrophilic proteins and membrane protein. (C) 2000 Elsevier Science B.V. All rights reserved.