Phase separation is a simple, efficient, and cheap method to purify and concentrate detergent-solubilized membrane proteins. In spite of this, phase separation is not widely used or even known among membrane protein scientists, and ready-to-use protocols are available for only relatively few detergent/membrane protein combinations. Here, we summarize the physical and chemical parameters that influence the phase separation behavior of detergents commonly used for membrane protein studies. Examples for the successful purification of membrane proteins using this method with different classes of detergents are provided. As the choice of the detergent is critical in many downstream applications (e.g., membrane protein crystallization or functional assays), we discuss how new phase separation protocols can be developed for a given detergent buffer system.
Detergents are used to extract membrane proteins from biological membranes and to mediate their solubility in aqueous solutions, which is a prerequisite for further protein purification (1). Purification of membrane proteins is generally tedious (2) because they are removed from their native membrane environment into a detergent buffer that can only partially mimick the physical and chemical properties of a lipid membrane. Thus, many membrane proteins do not retain their biological activity after extraction, or do so only partially or only under very special buffer conditions.
After extraction, purification of membrane proteins is usually accomplished by the same chromatography methods used for soluble proteins, the difference being that detergents must be present in the buffers at all times. Detergents do not interfere with ion exchange or metal chelate chromatography, and only sometimes with other affinity chromatography methods. In size exclusion chromatography, the apparent molecular weight of membrane proteins is increased by the bound detergent.
Phase separation is a powerful alternative or addition to chromatography-based purification protocols. It can be used directly on solubilized membranes, separating membrane proteins from soluble proteins and other hydrophilic impurities. Such crude purification protocols are used in membrane proteomics or as a first purification step followed by chromatography (3,4). Alternatively, phase separation can be exploited as a simultaneous concentration and polishing step at a later stage of purification (5).
Using phase separation steps in the purification of membrane proteins has a number of benefits. The protocols are simple to use, do not require complex laboratory equipment, are easily scaled up to large volumes, and are compatible with most chromatographic methods. Especially, the removal of hydrophilic compounds is very efficient. Moreover, membrane proteins can be simultaneously purified and concentrated, comparable in efficiency with precipitation protocols for soluble proteins that employ (NH4)2SO4 or other salts. A possible disadvantage of the method are the high detergent concentrations involved, which can be unfavorable for protein stability and can interfere with biochemical assays and binding processes. Dialysis or other methods like detergent absorption or gel filtration might be necessary to remove excess detergent from the solution.
Here, we describe the physical and chemical parameters that influence phase separation of the different classes of detergents commonly used in membrane protein purification. We discuss successful phase separation protocols and give guidelines as to how such protocols can be adapted to new detergent buffer systems.General Properties of Detergents
Detergents are amphipatic molecules usually consisting of a polar or charged headgroup and an extended hydrophobic hydrocarbon chain. At very low concentrations, these molecules are soluble in water as monomers. When increasing the detergent concentration above the so-called critical micelle concentration (CMC), the detergent molecules form aggregates with a very narrow size distribution, called micelles. The size of the micelles is dependent on the type of detergent; for detergents typically used in membrane biochemistry, the aggregation number (i.e., the number of detergent molecules per micelle) can range from 2 to 3 for sodium cholate to approximately 140 for Triton® X-100 (6). Moreover, micelle size and CMC depend on the ionic strength and the temperature of the detergent solution. While the CMC of ionic detergents decreases with increasing salt concentrations but is hardly affected by temperature, the CMC of nonionic detergents is only little affected by the presence of salts but increases with increasing temperature (7). Other factors that influence the size of the micelle and the CMC are pressure, pH, and the presence of impurities (6).Cloud Point and Phase Separation
The so-called cloud point can be reached by increasing the detergent concentration or by changing the temperature or the salt concentration of an aqueous micellar detergent solution. The micellar solution then becomes turbid; the micelles become immiscible with water and form large aggregates that will separate from the water phase. Most but not all of the detergent partitions into the detergent-rich phase, which in turn still contains a substantial amount of water. Depending on the detergent and the buffer conditions, the detergent-rich phase can be completely clear or turbid and can be found on top or below the detergent-poor phase. This process is called phase separation or, sometimes, cloud point extraction. Phase separation occurs due to a temperature-dependent difference in entropy between the one-phase and two-phase system. The effect is similar to protein precipitation using polyethelene glycol (PEG) or (NH4)2SO4 where not enough free water is available to keep the protein fully hydrated and thus, soluble. Likewise, the detergent micelles aggregate and form a separate phase in which less water covers their surface, and this aggregation behavior is influenced by temperature, salts, and polymers.