Separation methods in the analysis of protein membrane complexes.

The separation of membrane protein complexes can be divided into two categories. One category, which is operated on a relatively large scale, aims to purify the membrane protein complex from membrane fractions while retaining its native form, mainly to characterize its nature. The other category aims to analyze the constituents of the membrane protein complex, usually on a small scale. Both of these face the difficulty of isolating the membrane protein complex without interference originating from the hydrophobic nature of membrane proteins or from the close association with membrane lipids. To overcome this difficulty, many methods have been employed. Crystallized membrane protein complexes are the most successful example of the former category. In these purification methods, special efforts are made in the steps prior to the column chromatography to enrich the target membrane protein complexes. Although there are specific aspects for each complex, the most popular method for isolating these membrane protein complexes is anion-exchange column chromatography, especially using weak anion-exchange columns. Another remarkable trend is metal affinity column chromatography, which purifies the membrane protein complex as an intact complex in one step. Such protein complexes contain subunit proteins which are genetically engineered so as to include multiple-histidine tags at carboxyl- or amino-termini. The key to these successes for multi-subunit complex isolation is the idea of keeping the expression at its physiological level, rather than overexpression. On the other hand, affinity purification using the Fv fragment, in which a Strep tag is genetically introduced, is ideal because this method does not introduce any change to the target protein. These purification methods supported by affinity interaction can be applied to minor membrane protein complexes in the membrane system. Isoelectric focusing (IEF) and blue native (BN) electrophoresis have also been employed to prepare membrane protein complexes. Generally, a combination of two or more chromatographic and/or electrophoretic methods is conducted to separate membrane protein complexes. IEF or BN electrophoresis followed by 2nd dimension electrophoresis serve as useful tools for analytical demand. However, some problems still exist in the 2D electrophoresis using IEF. To resolve such problems, many attempts have been made, e.g. introduction of new chaotropes, surfactants, reductants or supporting matrices. This review will focus in particular on two topics: the preparative methods that achieved purification of membrane protein complexes in the native (intact) form, and the analytical methods oriented to resolve the membrane proteins. The characteristics of these purification and analytical methods will be discussed along with plausible future developments taking into account the nature of membrane protein complexes.