1.3 Recombinant
Expression
in Eukaryotic
Expression Systems
Expression of recombinant eukaryotic membrane protein in E. coli
frequently results in protein misfolding and aggregation. This can
be due to differences in the lipid composition, a different chaperone
repertoire, or the lack of post-translational modifications required
to obtain folded, active membrane protein. In these cases, eukaryotic expression systems that are more closely related to the natural
source of the protein have to be used. Unfortunately, membrane
protein expression in mammalian cells often results in moderate or
low protein yields. This is attributed to a limited capability of
mammalian transport systems to import additional proteins or to
replace its own proteins within the membranes of a particular
subcellular compartment. GFP fusion to the membrane protein of
interest is less useful for quality control and for optimizing expression in mammalian cells because in contrast to E. coli, the GFP will
fold independently from the membrane protein fused to it. Thus,
the GFP fusion method detects folded proteins as well as misfolded
proteins which are retained in the endoplasmic reticulum
membrane [17].
The majority of membrane proteins produced for structural
and functional analysis to date are from baculovirus insect cells
[18]. Baculovirus insect cell expression has been particularly successful for production of G protein-coupled receptors (GPCRs),
leading to the first crystal structures of this class of membrane
proteins [19, 20]. The MultiBac system [21] has the same modular
architecture of donor/acceptor vectors as described for the
ACEMBL system for E. coli expression above. This supports the
production of membrane protein complexes in insect cells, such as
the active γ-secretase complex comprised of four membrane protein
subunits [22].
1.4 Overexpression
in the Endogenous
Host
Isolation of membrane protein complexes from the natural host
used to be restricted to membrane protein machines which are
highly abundant in the cell, such as complexes for photosynthesis,
respiration, and oxidative phosphorylation [23]. However, with the
advent of miniaturization and automatization of biochemical, biophysical, and structural analyses, less abundant membrane protein
complexes can now be studied.
Purification of endogenous complexes is significantly facilitated
if a purification tag is fused to one of the subunits, which is possible
if the source can be genetically modified. The tagged subunit is
ideally an integral subunit of the complex. The chosen affinity
purification tag should be compatible with the presence of detergents in the buffer (frequent choices include His-tags, 3xFLAGtag, and CBP-tag).
The E. coli holo-translocon production described below uses
the endogenous host, thus providing the required chaperone repertoire and the physiological lipid composition. The purified complex has been shown to be active in co- and post-translational
Production of Multi-subunit Membrane Protein Complexes
7
Expression
in Eukaryotic
Expression Systems
Expression of recombinant eukaryotic membrane protein in E. coli
frequently results in protein misfolding and aggregation. This can
be due to differences in the lipid composition, a different chaperone
repertoire, or the lack of post-translational modifications required
to obtain folded, active membrane protein. In these cases, eukaryotic expression systems that are more closely related to the natural
source of the protein have to be used. Unfortunately, membrane
protein expression in mammalian cells often results in moderate or
low protein yields. This is attributed to a limited capability of
mammalian transport systems to import additional proteins or to
replace its own proteins within the membranes of a particular
subcellular compartment. GFP fusion to the membrane protein of
interest is less useful for quality control and for optimizing expression in mammalian cells because in contrast to E. coli, the GFP will
fold independently from the membrane protein fused to it. Thus,
the GFP fusion method detects folded proteins as well as misfolded
proteins which are retained in the endoplasmic reticulum
membrane [17].
The majority of membrane proteins produced for structural
and functional analysis to date are from baculovirus insect cells
[18]. Baculovirus insect cell expression has been particularly successful for production of G protein-coupled receptors (GPCRs),
leading to the first crystal structures of this class of membrane
proteins [19, 20]. The MultiBac system [21] has the same modular
architecture of donor/acceptor vectors as described for the
ACEMBL system for E. coli expression above. This supports the
production of membrane protein complexes in insect cells, such as
the active γ-secretase complex comprised of four membrane protein
subunits [22].
1.4 Overexpression
in the Endogenous
Host
Isolation of membrane protein complexes from the natural host
used to be restricted to membrane protein machines which are
highly abundant in the cell, such as complexes for photosynthesis,
respiration, and oxidative phosphorylation [23]. However, with the
advent of miniaturization and automatization of biochemical, biophysical, and structural analyses, less abundant membrane protein
complexes can now be studied.
Purification of endogenous complexes is significantly facilitated
if a purification tag is fused to one of the subunits, which is possible
if the source can be genetically modified. The tagged subunit is
ideally an integral subunit of the complex. The chosen affinity
purification tag should be compatible with the presence of detergents in the buffer (frequent choices include His-tags, 3xFLAGtag, and CBP-tag).
The E. coli holo-translocon production described below uses
the endogenous host, thus providing the required chaperone repertoire and the physiological lipid composition. The purified complex has been shown to be active in co- and post-translational
Production of Multi-subunit Membrane Protein Complexes
7