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 membrane proteins. Mechanisms for phase behaviour and protein partitioning of both membrane-bound and hydrophilic proteins have been examined in a large number of detergent/polymer aqueous two-phase systems. Non-ionic detergents such as the Triton series (polyoxyethylene alkyl phenols), alkyl polyoxyethylene ethers (C(m)EO(n)), Tween series (polyoxyethylene sorbitol esters) and alkylglucosides form aqueous two-phase systems in mixtures with hydrophilic polymers, such as PEG or dextran, at low and moderate temperatures. Phase diagrams for these mixtures are shown and phase behaviour is discussed from a thermodynamic model. Membrane proteins, such as bacteriorhodopsin and cholesterol oxidase, were partitioned strongly to the micelle phase, while hydrophilic proteins, BSA and lysozyme, were partitioned to the polymer phase. The partitioning of membrane protein is mainly determined by non-specific hydrophobic interactions between detergent and membrane protein. An increased partitioning of membrane proteins to the micelle phase was found with an increased detergent concentration difference between the phases, lower polymer molecular weight and increased micelle size. Partitioning of hydrophilic proteins is mainly related to excluded volume effects, i.e. increased phase component size made the hydrophilic proteins partition more to the opposite phase. Addition of ionic detergent to the system changed the partitioning of membrane proteins slightly, but had a strong effect on hydrophilic proteins, and can be used for enhanced separation between hydrophilic proteins and membrane protein.

Protein partitioning in weakly charged polymer-surfactant aqueous two-phase systems.

The study includes partitioning of proteins in aqueous two-phase systems consisting of the polymer dextran and the non-ionic surfactant C12E5 (pentaethylene glycol mono-n-dodecyl ether). In this system a micelle-enriched phase is in equilibrium with a polymer-enriched phase. Charges can be introduced into the micelles by the addition of charged surfactants. The charge of the mixed micelles is easily varied in sign and magnitude independently of pH, by the addition of different amounts of negatively charged surfactant, sodium dodecyl sulphate (SDS), or positively charged surfactant dodecyl trimethyl ammonium chloride (DoTAC). A series of water-soluble model proteins (BSA, beta-lactoglobulin, myoglobin, cytochrome c and lysozyme), with different net charges at pH 7.1, have been partitioned in non-charged systems and in systems with charged mixed micelles or charged polymer (dextran sulphate). It is shown that partition coefficients for charged proteins in dextran-C12E5 systems can be strongly affected by addition of charged surfactants (SDS, DoTAC) or polymer (dextran sulphate) and that the effects are directly correlated to protein net charge.

Affinity partitioning of a poly(histidine)-tagged integral membrane protein, cytochrome bo3 ubiquinol oxidase, in a detergent--polymer aqueous two-phase system containing metal-chelating polymer.

A system has been developed for selective partitioning of membrane proteins. For the first time, an integral membrane protein, cytochrome bo3 ubiquinol oxidase from Escherichia coli, has been affinity partitioned in an aqueous two-phase system. The systems used were different detergent/polymer aqueous two-phase systems containing a metal-chelating polymer, such as poly(ethyleneglycol)-iminodiacetic acid-Cu(II) as well as dextran-iminodiacetic acid-Cu(II). Many non-ionic detergents, such as alkyl(polyethyleneoxide) (CmEOn), Triton, Tween and alkylglucosides, form two-phase systems in mixture with polymers, such as dextran and poly(ethyleneglycol), i.e., a micelle-enriched phase in equilibrium with a polymer-enriched phase are formed. In general, membrane proteins partition strongly to the micelle phase. We show that it is possible to selectively partition a poly(histidine)-tagged integral membrane protein into the polymer phase by metal affinity partitioning, with a shift in the partitioning coefficient from 0.015 to 4.8 (300-fold). The affinity partitioning was characterized and the effects of ligand concentration, pH, time, salts, buffer type, imidazole and charged detergent are discussed. Thus, a fast and mild affinity procedure for the purification of integral membrane proteins can be developed in affinity detergent/polymer aqueous two-phase systems, and the method is especially suitable for the purification of labile integral membrane proteins, such as receptors.

Phase behavior and protein partitioning in aqueous two-phase systems of cationic--anionic surfactant mixtures.

Cationic-anionic surfactant mixtures can form aqueous two-phase systems. Such aqueous surfactant two-phase systems (ASTP systems) can be used for separation and purification of biomaterials. In this work we investigated the phase behavior and the partitioning of BSA and lysozyme in the ASTP system formed by mixtures of dodecyltriethylammonium bromide and sodium dodecylsulfate (SDS). The pseudo ternary phase diagram of these mixtures at low total surfactant concentrations contains two narrow two-phase regions, which represent two kinds of different ASTP systems formed when cationic and anionic surfactants are in excess, respectively (called ASTP-C and ASTP-A). The phase separation is associative, one phase is surfactant-rich, and the other phase is surfactant-depleted. Mechanisms behind the phase behavior are discussed. The phase behavior, especially phase separation time and phase volume ratio, is strongly influenced by total concentration and molar ratio of mixed surfactants. The effect of molar ratio is strong, which enables one to get desired phase systems also at very low total concentration by tuning the molar ratio of the surfactants. It was shown that the marked differences of surfactant concentration between the phases makes proteins distribute with different partitioning coefficients. The charges on the micellar surface, which can be adjusted by tuning the molar ratio of cationic surfactants to anionic surfactants, enhance the selectivity of protein partitioning by electrostatic effects. At pH 7.1, in the ASTP-C systems, negatively charged BSA is concentrated in the surfactant-rich phase and positively charged lysozyme in the surfactant-depleted phase, while in ASTP-A systems, a totally opposite partitioning was observed. It was shown that lysozyme could retain activity in ASTP systems.