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296 Lee and Aguilar
The primary difficulty encountered in the study of membrane proteins is
that of obtaining the protein of interest. The difficulties in the investigation
and separation of membrane proteins originate from their nature as membrane
proteins (1). Membrane proteins are usually present at very low levels in
biological membranes (2). They are very hydrophobic and have single or
several transmembrane parts, or closely associate with the membrane (3). In
the functional form, many of them comprise (homologous or heterologous)
multi-subunit complexes (4). Such membrane protein complexes contain many
cofactors and, inevitably, lipids (5). Some membrane protein complexes have
several peripheral proteins, which are functionally important but easily detached
during the isolation process. Despite the inherent difficulties of working with
membrane proteins, they remain an important area for study because of their
role in the control of fundamental biochemical process and their importance as
pharmaceutical targets (3).
In general, the methods available for the purification of membrane proteins
are basically the same as those employed to purify water-soluble, nonmembrane-associated
proteins (4–6). These methods include precipitation, gel
filtration, ion exchange, reversed phase, and affinity chromatography. Several
unique characteristics of membrane proteins, however, often make it difficult
to apply these methods successfully. It is important to stress that, just as
with soluble proteins, there is no way to present a single, precise set of
methods for the purification of all membrane proteins. Each membrane protein
possesses a unique set of physical characteristics, and conditions that are
suitable for the purification of one protein may not be suitable for others. As
a single chromatographic separation is not always successful in analyzing and
isolating the protein of interest, the combination of various modes of chromatography
is being developed for the study and separation of complex membrane
proteomes (7).
Owing to the hydrophobic nature and the complexity of proteins that reside
in biomembranes, immobilization of various modified phospholipids onto the
surface of chromatographic supports which potentially mimics the physicochemical
properties of biomembrane surfaces provides an additional dimension
in analyzing and separating membrane proteins (8–14). The chromatographic
supports modified with various phospholipid molecules, such as phosphatidylcholine,
phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine,
and phosphatidic acids, have been applied mainly for the analysis of drug–
membrane partition (15,16) and peptide–membrane interactions (10). However,
only columns packed with phosphatidylcholine-immobilized spherical particles
are commercially available, the structure of which is shown in Fig. 1.