JMMB
Symposium
Mdr Efflux
Proteins 193
J. Mol. Microbiol. Biotechnol. (2001) 3(2): 193-200.
Prokaryote Multidrug Efflux Proteins of the Major
Facilitator Superfamily: Amplified Expression,
Purification and Characterisation
Alison Ward1, Chris Hoyle2, Sarah Palmer3,
John O’Reilly1, Jeff Griffith4, Martin Pos1,5,
Scott Morrison1, Bert Poolman6, Mick Gwynne7,
and Peter Henderson1,*
1Astbury
Centre for Structural Molecular Biology, School
of Biochemistry and Molecular Biology, University of
Leeds, Leeds, LS2 9JT, UK.
2Novel Methods Group, SmithKline Beecham
Pharmaceuticals, New Frontiers Science Park (North),
Third Avenue, Harlow, Essex CM19 5AW, UK
3Celltech Chiroscience Ltd, 216 Bath Road, Slough,
SL1 4EN, UK
4Department of Cell Biology, UNM Cancer Research and
Treatment Center, 900 Camino de Salud NE,
Albuquerque, NM 87131, USA
5Institute of Microbiology, ETHZ LFV D18,
Schmelzbergstrasse 7, CH-8092 Zurich, Switzerland
6Department of Biochemistry, Groningen Biomolecular
Sciences and Biotechnology Institute, University of
Groningen, Nijenborgh 4, 9747 AG Groningen,
The Netherlands
7Anti-Infectives (UP1345), SmithKline Beecham
Pharmaceuticals, 1250 South Collegeville Road,
Collegeville, PA 19426-0989, USA
Introduction
Membrane transport proteins are involved in antibiotic
efflux, nutrient capture, environmental sensing, protein
secretion, toxin production, photosynthesis, oxidative
phosphorylation and other vital functions in bacteria (e.g.
Figure 1). Already there is commercial interest in inhibiting
the activities of some membrane transport proteins,
optimising the activities of others, employing them as
transducers of electrical/chemical/ mechanical energy for
nanotechnology, etc. However, membrane proteins are
notoriously difficult to study. Owing to their extreme
hydrophobicity they are refractory to direct manipulation
and can only be removed from the membrane, and their
solubility maintained, in the presence of detergent. In
addition membrane proteins are usually only expressed at
low levels and constitute less than 0.1% of total cell protein.
Such difficulties help to explain why, although the structures
of thousands of soluble proteins have been solved, to date
less than thirty membrane protein structures have been
resolved to atomic resolution (see examples in Sakai and
Tsukihara,1998; Wendt et al., 1999; Buchanan, 1999;
Abstract
In bacterial genomes 3-12% of open reading frames
are predicted to encode membrane transport proteins.
These proteins can be vital for antibiotic efflux, protein/
toxin secretion, cell nutrition, environmental sensing,
ATP synthesis, and other functions. Some, such as
the multidrug efflux proteins, are potential targets for
the development of new antibacterials and also for
applications in biotechnology. In general membrane
transport proteins are poorly understood, because of
the technical difficulties involved in isolating sufficient
protein for elucidation of their structure-activity
relationships. We describe a general strategy for the
amplified expression, purification and characterisation
of prokaryotic multidrug efflux proteins of the ‘Major
facilitator superfamily’ of transport proteins, using the
Bacillus subtilis multidrug resistance protein, ‘Bmr’,
as example.
*For correspondence. Email [email protected];
Tel. (44) 113 233 3122; Fax. (44) 113 233 3167.
© 2001 Horizon Scientific Press
Figure 1. Secondary active transport systems in bacteria. The large circle
represents the cytoplasmic membrane of the microorganism. A
transmembrane electrochemical gradient of protons is generated by
respiration or ATP hydrolysis, shown on the left. The gradient may be used
to drive ATP synthesis and the proton-nutrient symport and proton-substrate
antiport secondary active transport systems shown around the
circumference. Each is generally a single protein, usually of the 12-helix
type.
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194 Ward et al.
Table 1. Prokaryote MFS efflux transport proteins overexpressed in E. coli
Substrate(s)
Protein
Organism
Multidrugs
Multidrugs
Quinolones
Multidrugs?
Bicyclomycin
Tetracycline
aBmr
Bacillus subtilis
Bacillus subtilis
Staphylococus aureus
Methanococcus janaschii
Escherichia coli
Escherichia coli
a
b
aBlt
aNorA
aMj
1560
aBcr
bTetA
This work
Aldema et al. (1996)
Palczewski et al., 2000; Koronakis et al., 2000).
Analyses of the available bacterial genomes predict
that membrane transport proteins for the efflux of
‘multidrugs’ (Mdr’s) comprise 0.1-1.0% of the protein
complement (Paulsen et al., 2001). Further examination
of genomes from both prokaryote and eukaryote organisms
reveals that, like the majority of membrane transporters,
Mdr proteins can fall into two classes (Paulsen et al., 2001).
One of these utilises ATP to energise the transport of
substrates across the membrane - the ‘ATP-Binding
Cassette’ (ABC) superfamily, and the second, the ‘Major
Facilitator Superfamily’ or MFS (Saier et al., 2000) is usually
energised by the electrochemical gradient of protons in an
antiport mechanism (Figure 1). Members of the ABC
superfamily, and other types of Mdr efflux transport
systems, are considered elsewhere in this Symposium.
In bacteria individual MFS proteins may accomplish
the active accumulation of nutrient by a cation-substrate
symport mechanism (Figure 1), or the active efflux of
compounds like antibiotics, antibacterials, or toxins by a
cation/substrate antiport mechanism (Figure 1). MFS Mdr
proteins are thought to be single polypeptides comprising
10-14 (usually 12, Henderson, 1997) trans-membrane αhelices; this is illustrated for the Bacillus subtilis ‘Bmr’
protein (Neyfakh et al., 1991) in Figure 2. This conclusion
is usually based upon analysis of the hydropathic profile
of the amino acid sequence of each protein predicted from
its DNA sequence. In a few cases the prediction is
reinforced by genetic, immunochemical, or other types of
topological experiments, and in one case only, the Na+/H+
antiporter of Escherichia coli, NhaA, is there structural
information that confirms the 12-helix composition from
electron diffraction analyses of two-dimensional protein
crystals (Williams et al., 1999; Williams, 2000).
It is often the case that prokaryotic membrane proteins
have eukaryotic homologues, e.g. the prokaryotic MFS
multidrug efflux proteins are homologous to the vesicular
monoamine transport proteins that function in
neurotransmitter storage in nerve tissue (Yelin and
Schuldiner, 1995). Eukaryotic and prokaryotic transporters
can also share a very similar substrate profile although
they operate via a different mechanism eg: mammalian Pglycoprotein, Mdr, effluxes a similar range of compounds
as the Bacillus subtilis multidrug resistance protein, Bmr,
the Staphylococcus aureus norfloxacin resistance protein,
NorA and the Lactococcus lactis multidrug resistance
protein, LmrA (Neyfakh et al., 1991; van Veen et al., 1998).
The overexpression and characterization of prokaryotic
membrane transport proteins may, therefore, lead us to a
greater understanding of eukaryotic protein function. This
avoids the problems associated with eukaryotic expression
systems, such as incorrect post-translational modification
and transience of expression.
To enable the determination of structures of membrane
transport proteins a continuing supply of milligram
quantities of protein is required. As native expression levels
are usually less than 0.1% of total cell protein so genetical
amplification of expression must be developed. Even if such
amplification is successful a suitable detergent must be
found for purification. The protein may also require
‘conformation locking’ to overcome the probable flexibility
of transport proteins, which is invoked to account for their
ability to bind substrate in, or on one side of, the membrane,
and effect its translocation. The strategy needed to purify
sufficient protein is therefore complex.
In our laboratory a general strategy has been devised
for the amplified expression, purification and
characterisation of bacterial MFS membrane transport
proteins (Table 1) in Escherichia coli. The strategy is
described in this article to facilitate future examination of
the large number of MFS efflux proteins arising from
genome analyses that have potential for development of
novel antibacterials, and perhaps applications in
biotechnology. So far the strategy has been successful for
eighteen prokaryote transporters, including five Mdr
proteins from Bacillus subtilis, Staphylococcus aureus,
Methanococcus janaschii, and E. coli. Other organisms that
appear to be successful for the propagation of vectors and
expression of heterologous membrane proteins are
Table 2. Some expression systems for amplification of MFS Mdr prokaryote membrane transport proteins in E. coli
Promoter
Repressor
Inducer
Proteins expressed
E. coli host
Comments
tac
Lac
IPTG
E. coli sugar-H+ symporters; araE, gusB, xylE and fucP.
B. subtilis (bmr) and S. aureus (norA) multidrug efflux
proteins. See also Henstra et al., 1996; Kanamari et al.,
1999; Pourcher et al., 1995.
NO2947 NM554
Expression of up to 20% inner
membrane protein.tac promoter is not
tightly regulated and expression occurs
in the absence of inducer. tac is also E.
coli host strain independent.
T7
T7 lysozyme
IPTG
E. coli tetracycline H+ antiporter (Aldema et al., 1996).
BL21(DE3) and its
mutant forms DH5α
with pACT7
Usually generates inclusion bodies.
Expression occurs when plasmids are
placed in hosts producing T7 RNA
polymerase (usually under the control of
the lacUV promoter, which is IPTG
inducible). The lacUV promoter is not
tightly controlled, but transformation into
BL21(DE3)plysS or plysE produces T7
lysozyme which inhibits T7 RNA
polymerase.
Mdr Efflux Proteins 195
Figure 2. A two-dimensional model for the folding of the Bmr multidrug efflux protein in the cell membrane
Lactococcus lactis and Streptococcus thermophilus
(Poolman, 2000) and Halobacterium salinarum (Turner et
al., 1999).
E. coli Expression Systems: Successes and Failures
To facilitate the study of prokaryotic membrane proteins
numerous E. coli expression systems have been used
[reviewed in Grisshammer and Tate, 1995; Table 2] with
levels of expression being reported of up to 50% inner
membrane protein (Ward et al., 2000) and 80% outer
membrane protein (Ghosh et al., 1998).
We have successfully overexpressed eighteen
prokaryotic membrane transport proteins in E. coli, with
expression levels ranging from 20-50% of inner membrane
Figure 3. The plasmid pTTQ18 (Stark, 1987) cloning vector for the amplified
expression of membrane transport proteins in E. coli. Restriction enzyme
sites, especially in the multiple cloning site, are illustrated.
protein. Most of these proteins are predicted, by hydropathy
analysis of their amino acid sequence, to comprise twelve
transmembrane spanning α-helices with the N- and Ctermini located in the cytoplasm (Henderson, 1997; Figure
2). Sixteen of the membrane transport proteins, including
those for efflux of drugs/antibiotics and for the uptake of
nutrients (sugars, amino acids), have been amplified to
approximately 20-30% inner membrane protein, using the
plasmid pTTQ18 (Stark, 1987; Table 2; Figure 3).
Transcription from plasmid pTTQ18 is directed from the
tac promoter, which in the absence of IPTG is repressed
by the plasmid encoded lac repressor (Figure 2; Ward et
al., 2000 for details). The gene for each membrane protein
is ligated downstream of the tac promoter, and an
oligonucleotide encoding six histidines and, usually, an
epitope tag is added, in-frame, to the C-terminus (amino
acid sequence -G-G-R-G-S-H-H-H-H-H-H, Hoyle,
unpublished, and see below). The N-terminal amino acid
sequence of the membrane protein may be slightly modified
by a few amino acids from the α-peptide of the LacZ protein,
which is in the multicloning site, but this does not prevent
expression (Hoyle, unpublished). Growth conditions are
optimised in 1-25 litre cultures of E. coli host strains, testing
both minimal and complex media to maximise expression
of each protein (Ward et al., 2000). The concentration of
IPTG required is tested between 0.1-1.0mM, and the period
of growth before induction varied to obtain as high a cell
density as possible commensurate with optimal protein
expression (growth is often diminished, or abolished, after
induction – see below). Similarly, the period of exposure
to IPTG (2-24 hours) is investigated in order to promote
maximum expression and minimise the following problem.
Perhaps the most common difficulty associated with
membrane protein expression is toxicity and a reduction
in host cell viability post induction. For example, whilst the
overexpression of the E. coli sugar transporters does not
have a deleterious effect on cell growth, the expression in
E. coli of both the Bacillus subtilis multidrug resistance
protein (Bmr) and the norfloxacin resistance protein (NorA)
196 Ward et al.
Figure 4. Prokaryotic membrane proteins overexpressed in E. coli. Samples
(30µg) of inner membrane protein, containing the overexpressed
hexahistidine tagged protein of interest, were separated on a 15%
polyacrylamide SDS-PAGE gel and stained with Coomassie Brilliant Blue.
Overexpressed proteins are indicated by arrow heads: track 1 shows Mr
markers; track 2 the E. coli galactose-H+ symporter, GalP, overexpressed
from the vector pBR322 under the control of its own promoter in the host
strain JM1100; track 3 the glucuronide-H+ symporter, GusB, from E. coli
overexpressed from the vector pTTQ18 under the control of the tac promoter
in the host strain NO2947; track 4 the fucose-H+ symporter, FucP, from E.
coli overexpressed from the vector pTTQ18 under the control of the tac
promoter in the host strain Blr; track 5 the multidrug efflux protein, Bmr,
from Bacillus subtilis overexpressed from the vector pTTQ18 under the
control of the tac promoter in the host strain Blr; track 6 the norfloxacin
resistance protein, NorA, from Staphylococcus aureus, which shares 44%
amino acid identity with Bmr, overexpressed from the vector pTTQ18 under
the control of the tac promoter in the host strain Blr.
from Staphylococcus aureus results in a rapid attenuation
of cell growth and ultimately in cell death. Cells expressing
Bmr can lyse after only a few hours (Ward and Hoyle,
unpublished); cells over-expressing NorA have impaired
cell division and elongate (Hoyle, unpublished); and cells
over-expressing TetA undergo a rapid loss of membrane
potential similar to that observed on the addition of
uncoupling agents (Eckert and Beck, 1989). One or more
of these problems is often more evident with Mdr proteins
than other members of the MFS family, in our experience.
Other bacterial multidrug transporters are also toxic
(Poolman, personal communication), so these examples
illustrate that toxicity is often related to the particular
function of the membrane protein. In such cases
overexpression can be achieved by the careful choice of
media, host strain, growth temperature and titration of
inducer (Kanamari et al., 1999). Miroux and Walker (1996)
have described mutants of BL21(DE3) host strains for the
successful expression of membrane proteins with the
avoidance of toxicity, although in some cases the protein
of interest accumulated as inclusion bodies. Inclusion
bodies generate extremely large quantities of protein (up
to grams per litre of culture), albeit in an insoluble form.
Whilst there are no general methods available for the
refolding of polytopic membrane protein inclusion bodies,
recent work has demonstrated the refolding of hexahistidine
tagged membrane proteins using nickel chelate affinity
chromatography in conjunction with a number of detergent
wash steps (Rogl et al.,1998; Anderson et al., 1999;
Buchanan, 1999)
In many cases a 25l fermenter can conveniently be
used without compromising expression, but for some
proteins the level of expression is always higher in batch
cultures of 500-800ml in 2l baffled flasks. Further
examination of the parameters regulating growth and
protein production in these conditions may enhance our
understanding of expression.
It is possible that the cell’s machinery for folding and
insertion of transport proteins into the membrane, which
require multiple factors in E. coli, may limit overexpression.
Studies with the E. coli lactose transporter have shown
that the folding and insertion of this protein into the
membrane requires phosphatidylethanolamine (Bogdanov
and Dowhan 1998), the chaperonin GroEL (Bochkareva
et al., 1996), and the bacterial signal recognition particle
(MacFarlane and Muller, 1995). Growth at lower
temperatures, or using lower [IPTG] to slow expression,
may alleviate these effects.
The tac promoter has also been successfully employed
for the overexpression in E. coli of the Enzyme II mannitol
transport protein from the thermophilic Bacillus
stearothermophilis (Henstra et al., 1996) and the alanine
carrier from the thermophilic bacterium PS3 (Kanamari et
al., 1999) (as an N-terminal maltose binding protein fusion)
and the E. coli melibiose transporter (Pourcher et al.,1995).
The pET system (Novagen), which is widely used for
the expression of soluble proteins, has also led to the
amplified production of both native membrane protein
(Table 2; Pourcher et al., 1990; Aldema et al., 1996) and
inclusion bodies (Miroux and Walker, 1996). In pET vectors
the strong bacteriophage T7 promoter is recognised by
T7, but not E. coli, RNA polymerase. Expression from pET
vectors is achieved by transforming the recombinant
plasmid into a host strain which carries a chromosomal
copy of T7 RNA polymerase. For the usual E. coli (DE3)
lysogen host strains the T7 RNA polymerase is under the
control of the lacUV5 promoter (Studier et al., 1990),
thereby allowing some expression of T7 RNA polymerase
even in the absence of inducer.
Identification of Over-Expressed Membrane Proteins
The success of the amplified expression is usually first
indicated by the appearance of an extra protein in
preparations of inner membranes stained with Coomassie
Blue or silver in SDS-PAGE gels (Figure 4). The levels of
expression of Bmr(His)6 and NorA(His)6 are comparable
to those achieved for sugar transport proteins (Figure 4).
It is very important to note two things: membrane proteins
rarely solubilise well when boiled in SDS, unlike soluble
proteins; also, all MFS proteins in our experience migrate
at apparent Mr values below those predicted from their
amino acid sequence. Therefore, lower temperatures must
be used for solubilisation (Ward et al., 2000). It is then
very advisable to confirm the identity, and the absence of
post-translational modification, by eluting the protein and
carrying out N-terminal amino acid sequencing and by
using Western blotting to ensure that the C-terminal Histag is present (Ward et al., 2000). A variable phenomenon,
in our experience, is the appearance, in stained SDS-PAGE
gels and Western blots, of apparently higher molecular
weight forms of the same protein; an example is shown for
Bmr(His)6 in Figure 5. If this represents some aggregation
of the protein, then steps may be needed to eliminate it
Mdr Efflux Proteins 197
Figure 5. The purification of Bmr(His)6 from inner membranes of E. coli
using Ni-NTA affinity chromatography A. Silver stained 15% SDS-PAGE
gel. Track 1, molecular weight markers (‘Mr ’). Inner membranes (‘IM’, track
2; Ward et al., 2000), 20 mg, were solubilised in 100mM Hepes buffer, pH
7.9, 20% glycerol, 1% dodecyl-β-D-maltoside (DDM), 20mM imidazole
(buffer E), while stirring on ice for 30 min. The treated membranes were
centrifuged at 100,000 g for 30 min at 4°C to remove unsolubilised material
and the supernatant (‘+DDM’, track 3) was mixed with 1 ml Ni-NTA resin
after equilibration in buffer E, and incubated with gentle shaking overnight
(12-16h) at 4°C. The resin was collected by centrifugation, 198g 1min at
room temperature and the supernatant retained (‘Unbound’ , track 4). The
protein-resin complex was packed into a disposable column and the
unbound protein was further removed by washing the resin with 25ml buffer
E containing 0.05% DDM, pH 8 (‘Wash’, track 5) Finally, Bmr(His)6 was
eluted from the column with 5ml buffer E containing containing 0.05% DDM
and 200mM imidazole pH8, and collected in fractions; those fractions
containing protein were concentrated (‘Eluate’, track 6). The tendency of
some of the transport proteins to reveal an apparently higher Mr form
(oligomer/conformer?) is illustrated in this case. The bmr gene was kindly
provided by A. Neyfakh.
before crystallisation trials. Alternatively, it may represent
different conformers of the fully/partially unfolded protein,
even in SDS.
Recently, protocols have been devised to enable MFS
proteins to be analysed in mass spectrometers, so
determining the precise Mr of purified material (Ward et
al., 2000; le Coutre et al., 2000). Partial proteolytic digestion
followed by MS-MS amino acid sequencing can then
reinforce identification.
Purification of Membrane Proteins Facilitated by the
Addition of Affinity Tags
Whilst there are a variety of affinity fusions (Nilsson et al.,
1997) available for the addition to membrane proteins to
facilitate their purification, perhaps the most widely used
is hexahistidine. The addition of a hexahistidine tag to the
membrane protein of interest allows its rapid purification
using nickel chelate affinity chromatography (Pos et al.,
1994; Pourcher et al.,1995). This tag is small and should
not decrease expression levels or have a deleterious effect
on protein folding or insertion into the membrane.
Experience indicates that the hexahistidine tag is in itself
not a barrier to crystallization (e.g. Zhang et al., 1994), but
if it is desirable to remove the hexahistidine tag after protein
purification, the genetic insertion of a suitable protease
cleavage site, such as thrombin, readily allows this. An
additional advantage to the use of such tags is that often
antibodies to the tags are commercially available, so
facilitating Western blotting for protein detection without
the need to raise antibodies against the protein of interest.
Using nickel chelate affinity chromatography we have
routinely purified proteins to greater than 90% homogeneity,
as judged by densitometry (e.g. Figure 5).
In order to aid crystallisation, we have also fused the
gene encoding Bmr(His)6 or NorA(His)6 in-frame to ‘green
fluorescent protein’. In these two examples a chimaeric
protein was successfully produced, but the amplification
of expression was severely attenuated. When the fusion
was made of MalE (maltose binding protein, MBP, lacking
signal peptide so it was not exported) to the N-terminus of
Bmr, the chimaeric protein was expressed at levels
sufficient for structural studies. In all cases the attached (His)6 tag was used to effect purification, though the
additional MBP protein also allows purification using
amylose resin. This may require exchange of detergent,
from dodecyl-β-D-maltoside to Triton X-100, for example.
Choice of Detergent and Solubilisation Conditions
Integral membrane proteins are removed from lipid bilayers
by the action of detergents. Detergents are amphipathic
molecules comprising a polar head group and a
hydrocarbon tail. At a determined concentration, referred
to as the critical micelle concentration (CMC) detergent
monomers aggregate to form ordered structures into which
membrane proteins can insert. Detergents are classified
into three broad categories:
• ionic, which carry a net charge associated with their
head group eg: the anionic sodium dodecyl sulphate
• non-ionic, which have uncharged hydrophilic head
groups eg: dodecyl-ß-D-maltoside
• zwitterionic, which have both positive and negative
charges but carry no net charge eg: CHAPS
Detergents used in the solubilization and purification of
membrane proteins must maintain the structural integrity
of the protein and its activity on reconstitution. In many
cases it may not be possible to select a detergent suitable
for both solubilization and purification. In such cases
detergent exchange may be carried out (see Ward et al.,
2000). The solubilization of membrane proteins is a
multistep process. Below the CMC detergent monomers
partition into the lipid bilayer. As the concentration of the
detergent increases to CMC and above the membrane
breaks down to generate mixed micelles of protein/
detergent, protein/detergent/lipid, lipid/detergent and
detergent alone. Alternatively, a mixture of detergents used
below their CMC values may be effective (Koronakis et
al., 2000).
Membrane protein solubilization trials should be carried
out using a wide range of detergents at concentrations
ranging from CMC, protein concentrations in the range 110 mg/ml and in the presence of stabilizing additives such
as glycerol and NaCl. The solubilization mix is incubated
(usually on ice) before recovering the solubilized material
by ultracentrifugation. Individual membrane proteins will
show different solubilization requirements, especially of pH
and salt concentration. This property can sometimes be
used to pre-extract unwanted membrane proteins with one
detergent and subsequently solubilise the protein of interest
with a second detergent (e.g. Fang et al., 1999). The
process of solubilization can be monitored using SDSPAGE and western blotting.
Detailed protocols for optimising solubilisation of
198 Ward et al.
membrane proteins are given elsewhere (Ward et al.,
2000). For the Mdr proteins dodecyl-β-D-maltoside (DDM)
or cyclohexyl-n-hexyl-β-D-maltoside have proved to be the
detergent of choice for solubilisation and purification.
However, the best concentration of detergent and stabilising
additives (e.g. benzamidine, glycerol, sulphobetaine) still
needs to be determined for each protein.
Reconstitution and Assay of Membrane Protein Activity
There are two basic methods for the reconstitution of
detergent solubilized membrane symporters. In the first
membrane protein is mixed with detergent-destabilized
liposomes, rapidly diluted (to below the CMC of the
detergent) and the proteoliposomes recovered by
ultracentrifugation (Henderson et al.,1983). In the second
method protein is mixed with detergent-destabilized
liposomes and the detergent removed by successive
additions of polystyrene beads (Knol et al ., 1996)
(BiobeadsTM, Biorad): one or more dialysis steps may also
be required (Racher et al., 1999).
Activity of the reconstituted protein can be assayed
using artificial ion gradient-driven uptake (Jung et al., 1998),
enzyme-linked spectroscopic assays (Heuberger et al.,
2000), or possibly ligand binding assays. For artificial ion
gradient-driven studies proteoliposomes can be preloaded
with 100mM potassium acetate and 25mM potassium
phosphate, pH7.0. The reaction is initiated by dilution of
the proteoliposomes into 125mM sodium phosphate plus
the potassium ionophore valinomycin and radioactive
substrate. In this system acetic acid passively diffuses
across the proteoliposome membrane and then
dissociates, thereby removing protons from the vesicle
lumen and thus increasing the internal pH and transmembrane ∆pH. The addition of valinomycin causes a flux
of potassium, from the inside to the outside of the
proteoliposome, thereby generating a trans-membrane
potential (∆ϕ) (see, e.g. Racher et al., 1999). For Mdr
antiporters similar methods have been employed (Putman
et al., 1999).
Physical Measurements of Detergent Solubilized and
Reconstituted Membrane Protein
to be heavily biased towards soluble protein structure, and
so anomalous fitting results can occur.
FTIR spectra of proteins arise from peptide C=O
stretching vibrations. Two bands are evident, amide I and
amide II (Haris and Chapman, 1995).The deconvolution
of amide I separates the individual subcomponents of the
peak, and from the wavenumbers and intensities of these
deconvoluted peaks, secondary structure assignments can
be made. For example the peak for α-helices is found in
the region 1650-1658 cm-1. Unlike CD this method is not
affected by light scattering and is independent of protein
concentration. With all the Mdr proteins we have isolated
so far (Table 2), both CD and FTIR measurements indicate
a high content of α-helix (50-80%), which is tenaciously
retained in the detergent-solubilised state.
To gain structural information at atomic resolution, xray or electron crystallography of three and two dimensional
crystals, respectively, is required. Three dimensional
crystals are most commonly grown using the vapour
diffusion method (Howard et al ., 1999). Detergent
solubilized protein is mixed on a coverslip (usually at a 1:1
ratio) with precipitant, the coverslip is then inverted over a
well containing precipitant alone and the system allowed
to reach a state of supersaturation. To date, whilst 3D
crystals of different MFS membrane transport proteins have
been successfully grown, none have yielded high resolution
diffraction data. Two dimensional crystals are obtained
when detergent solubilized protein is mixed with lipiddetergent mixed micelles, after which the detergent is
removed by dialysis or adsorption onto polystyrene beads
(BiobeadsTM, Biorad) (Ringler et al., 1999). Ratios of lipid
to protein (weight:weight) are restricted so as to encourage
close packing of the protein into lipid bilayers when the
detergent is removed. 2D crystals of the E. coli melibioseNa+ and lactose-H+ transporters (Rigaud et al.,1997;
Zhuang et al., 1999), and the B. subtilis Bmr efflux protein
(Ward, Stolyova, Holzenburg and Henderson, unpublished)
have been grown, but ordering of the molecules is too low
to determine the structure of these proteins at high
resolution. The recent success with the sodium/H +
antiporter (Williams et al., 1999; Williams, 2000), however,
generates cautious optimism.
Conclusions
There are two methods which give an indication of a
protein’s secondary structure content, Circular Dichroism
(CD) and Fourier Transform Infra Red (FTIR) spectroscopy.
Both methods can be applied to both detergent solubilized
and membrane-reconstituted protein. For FTIR
spectroscopy exchange of H2O by D2O is usually carried
out to minimise errors caused by imprecise correction for
IR absorbance by the former (Patzlaff et al.,1998)
CD signals are generated when a molecule
differentially absorbs left and right handed circularly
polarized light. Characteristic spectra occur for each type
of secondary structure eg: α-helix has a positive peak at
195nm and two negative peaks at 208 and 222nm with an
indent between these two negative peaks at 215nm (Kelly
et al., 1997). CD can be affected by light scattering,
especially from proteoliposome solutions (Chin, 1986) and
interpretation is dependent upon an accurate estimate of
protein concentration. Algorithms for fitting CD data tend
The strategies described above are achieving amplified
expression of active prokaryotic Mdr transport proteins.
Furthermore, the levels reached are sufficient for
purification, characterisation and crystallisation trials. The
majority of successes have been achieved using the tac
promoter, for induction with IPTG, in various E. coli host
strains. However, other plasmids such as pBluescript and
the use of the galP promoter in the plasmid pBR322 have
been successful. To date, whilst the pET vector has been
used for the expression of membrane proteins as inclusion
bodies, this T7 promoter based plasmid has yet to find
widespread use for the overexpression of membrane
proteins directed to the membrane; nevertheless,
expression of membrane proteins as inclusion bodies may
prove a viable option with recently published protocols for
the refolding of membrane protein inclusion bodies (e.g.
Buchanan, 1999).
Mdr Efflux Proteins 199
The most commonly used tag employed for the affinity
purification of membrane proteins involves the addition of
hexahistidine to the C-terminus of the protein to facilitate
purification using nickel (or cobalt) chelate affinity
chromatography. This tag is small and has been shown
not to interfere with levels of overexpression achieved or
the targetting or folding of the protein. Moreover the
structure determination of soluble hexahistidine tagged
proteins suggests that the histidine tag is itself not a barrier
to crystallization.
In conclusion, with the careful choice of vector, E. coli
host strain and expression conditions, it is now possible to
overexpress prokaryotic Mdr membrane transport proteins
to produce mg quantities of pure protein per litre of culture.
This should pave the way for the elucidation of increasing
numbers of prokaryotic membrane transport protein
structures and ultimately the determination of their mode
of action at the molecular level.
Acknowledgements
We are grateful to the BBSRC, MRC, SmithKline Beecham Pharmaceuticals,
EU and Wellcome Trust for financial support. This work was carried out as
part of the BBSRC funded North of England Structural Biology Consortium.
The Biochemical Society and Academic Press kindly gave permission to
reproduce some material in Tables 1,2 and Figure 4.
References
Aldema, M.L., McMurry, L.M., Walmsley, A.R. and Levy, S.B. 1996.
Purification of the Tn 10-specified tetracycline efflux antiporter TetA in a
native state as a polyhistidine fusion protein. Molec. Microbiol. 19: 187195.
Anderson, M., Blowers, D., Hewitt, N., Hedge, P., Breeze, A., Hampton, I.
and Taylor, L. 1999. Refolding, purification, and characterization of a loop
deletion mutant of human Bcl-2 from bacterial inclusion bodies. Protein
Expression and Purification. 15: 162-170.
Bochkareva, E., Seluanov, A., Bibi, E. and Girshovich, A. 1996. Chaperoninpromoted post-translational membrane insertion of a multispanning
membrane protein lactose permease. J. Biol. Chem. 271: 22256-22261.
Bogdanov, M. and Dowhan, W. 1998. Phospholipid-assisted protein folding:
phosphatidylethanolamine is required at a late step of the conformational
maturation of the polytopic membrane protein lactose permease. EMBO
J. 17: 5255-5264.
Buchanan, S.K. 1999. ß-Barrel proteins from bacterial outer membranes:
structure, function and refolding. Curr. Opin. Struct. Biol. 9: 455-461
Chin, J.J., Jung, K.Y. and Jung, C.Y. 1986. Structural basis of human
erythrocyte glucose transporter function in reconstituted vesicles - alphahelix orientation. J. Biol. Chem 261: 7101-7104.
Eckert, B. and Beck, C.F. 1989. Overproduction of Transposon Tn10encoded tetracycline resistance protein results in cell-death and loss of
membrane-potential. J. Bacteriol. 171: 3557-3559.
Fang, G., Friesen, R., Lanfermeijer, F., Hagting, A., Poolman, B. and
Konings, W.N. 1999. Manipulation of activity and orientation of membranereconstituted di-tripeptide transport protein DtpT of Lactococcus lactis.
Molec. Memb. Biol. 16: 297-304.
Ghosh, R., Steiert, M., Hardmeyer, A., Wang, Y.F. and Rosenbusch, J.P.
1998. Overexpression of outer membrane porins in E. coli using
pBluescript-derived vectors. Gene Expression 7: 149-161
Grisshammer, R. and Tate, C.G. 1995. Overexpression of integral
membrane-proteins for structural studies. Q. Rev. Biophys. 28: 315-422.
Gunn, F.J., Tate, C.G.and Henderson, P.J.F. 1994. Identification of a novel
sugar-H+ symport protein, FucP, for transport of L-fucose into Escherichia
coli. Mol. Microbiol. 12: 799-809.
Haris, P.I. and Chapman, D. 1995. The conformational-analysis of peptides
using Fourier-transform IR spectroscopy. Biopolymers, 37: 251-263.
Henderson, P.J.F., Kagawa, Y. and Hirata, H. 1983. Reconstitution of the
GalP galactose transport activity of Escherichia coli into liposomes made
from soybean phospholipids. Biochim. Biophys. Acta. 732: 204-209.
Henderson, P.J.F. 1997. Function and structure of membrane transport
proteins in The Transporter Factsbook (Eds. Griffith, J.K. and Sansom,
C.) pp. 3-29. Academic Press, Oxford
Henstra, S.A., Tolner, B., Duurkens, R.H.T, Konings, W.N. and Robillard,
G.T. 1996. Cloning, expression, and isolation of the mannitol transport
protein from the thermophilic bacterium Bacillus stearothermophilus. J.
Bacteriol. 178: 5586-5591.
Heuberger, E.H.M.L. and Poolman, B. 2000. A spectroscopic assay for the
analysis of carbohydrate transport reactions. Eur. J. Biochem. 267: 228234.
Howard, T.D., McAulay-Hewcht, K.E. and Cogdell, R.J. 2000. Crystallisation
of membrane proteins. In ‘Membrane Transport - a Practical Approach’.
Baldwin, S.A., ed. Chapter 11, pp269-307. Blackwell’s Press :Oxford UK.
Jung, H., Tebbe, S., Schmid, R. and Jung, K. 1998. Unidirectional
reconstitution and characterization of purified Na+/proline transporter of
Escherichia coli. Biochemistry. 37: 11083-11088.
Knol, J., Veenhoff, L., Liang, W.J., Henderson, P.J.F., Leblanc. G. and
Poolman, B. 1996. Unidirectional reconstitution into detergent-destabilized
liposomes of the purified lactose transport system of Streptococcus
thermophilus. J. Biol. Chem. 271: 15358-15366.
Koronakis, V., Sharrff, A. Koronakis, E., Luisi, B., and Hughes, C. 2000.
Crystal structure of the bacterial outer membrane protein TolC central to
multidrug efflux and protein export. Nature 405, 914-919.
Le Coutre, J., Whitelegge, J.P., Gross, A., Turk, E., Wright, E.M., Kaback,
H.R. and Faull, K.F. 2000. Proteomics on full-length membrane proteins
using mass spectrometry. Biochemistry 39, 4237-4242.
Macfarlane, J. and Muller, M. 1995. The functional-integration of a polytopic
membrane-protein of Escherichia coli is dependent on the bacterial signalrecognition particle. Eur. J. Biochem. 233: 766-771.
Miroux, B. and Walker, J.E. 1996. Over-production of proteins in Escherichia
coli: mutant hosts that allow synthesis of some membrane proteins and
globular proteins at high levels. J. Mol. Biol. 260: 289-298.
Neyfakh, A.A., Bidnenko, V.E. and Chen, L.B. 1991. Efflux-mediated
multidrug resistance in Bacillus subtilis - similarities and dissimilarities
with the mammalian system. Proc. Natl. Acad. Sci. USA. 88: 4781-4785.
Nilsson, J., Stahl, S., Lundeberg, J., Uhlen, M. and Nygren, P.A. 1997.
Affinity fusion strategies for detection, purification, and immobilization of
recombinant proteins. Protein Expression and Purification. 11: 1-16.
Palczewski, K., Kuimasaka, T., Hori, T., Behnke, C.A., Motoshima, H., Fox,
B.A., Le Trong, I., Teller, D.C., Okada, T., Stenkamp, R.E., Yamamoto, M.
and Miyano, M. 2000. Science 289: 739- 745.
Patzlaff, J.S., Moeller, J.A., Barry, B.A., and Brooker, R.J. 1998. Fourier
transform infrared analysis of purified lactose permease: a monodisperse
lactose permease preparation is stably folded, alpha-helical, and highly
accessible to deuterium exchange. Biochemistry. 37: 15363-15375.
Paulsen, I.T., Chen, J., Nelson, K.E., and Saier, M.H.Jr. 2001. Comparative
genomics of microbial drug efflux systems. J. Mol. Microbiol. Biotech. 3:
145-150.
Poolman, B. , Fang, G., Friesen, R., Gunnewijk, M.G.W., van der Heide, T.,
Heuberger, E.H.M.L., Klunder, B., Knol, J. and Veenhoff, L.M. 2000.
Amplified gene expression in Gram-positive bacteria, and membrane
reconstitution of purified membrane transport proteins. In ‘Membrane
Transport - a Practical Approach’. Findlay, J.B.C., Harrison, M.A. and
Evans, W.H., ed. Blackwell’s Press: Oxford UK. In press.
Pos, K.M., Bott, M. and Dimroth, P. 1994. Purification of 2 active fusion
proteins of the Na+-dependent citrate carrier of Klebsiella pneumoniae.
FEBS Lett. 347: 37-41.
Pourcher, T., Bassilana, M., Sarkar, H.K., Kaback, H.R. and Leblanc, G.
1990. Melibiose permease and alpha-galactosidase of Escherichia coli identification by selective labeling using A T7 RNA-polymerase promoter
expression system. Biochemistry 29: 690-696.
Pourcher, T., Leclercq, S., Brandolin, G. and Leblanc, G. 1995. Melibiose
permease of Escherichia coli - Large-scale purification and evidence that
H+, Na+, And Li+ sugar symport is catalyzed by a single polypeptide.
Biochemistry 34: 4412-4420.
Putman, M., van Veen, H.W., Poolman, B. and Konings, W.N. 1999.
Restrictive use of detergents in the functional reconstitution of the
secondary multidrug transporter LmrP. Biochemistry. 38: 1002-1008.
Racher, K.I., Voegele, R.T., Marshall, E.V., Culham, D.E., Wood, J.M., Jung,
H., Bacon, M., Cairns, M.T., Ferguson, S.M., Liang, W-j., Henderson,
P.J.F., White, G. and Hallett, F.R. 1999. Purification and reconstitution of
an osmosensor: transporter ProP of Escherichia coli senses and responds
to osmotic shifts. Biochemistry 38: 1676-1684
Rigaud, J.L., Mosser, G., Lacapere, J.J., Olofsson, A., Levy, D. and Ranck,
J.L. 1997. Bio-beads: an efficient strategy for two-dimensional
crystallization of membrane proteins. J. Struc. Biol. 118: 226-235.
Ringler, P., Heymann, B. and Engel, A. 2000. Two-dimensional crystallisation
of membrane proteins. In ‘Membrane Transport - a Practical Approach’.
Baldwin, S.A., ed. Chapter 10, pp229-268. Blackwell’s Press :Oxford UK.
Rogl, H., Kosemund, K., Kuhlbrandt, W. and Collinson, I. 1998. Refolding
of Escherichia coli produced membrane protein inclusion bodies
immobilised by nickel chelating chromatography. FEBS Letters. 432: 2126.
Sakai, H. and Tsukihara, T. 1998. Structures of membrane proteins
determined at atomic resolution. J. Biochem. 124: 1051-1059.
Saier , M.H., Beatty, J.T., Goffeau, A., Harley, K.T., Heijne, W.H.M., Huang,
200 Ward et al.
S.-C., Jack, D.L., Jahn, P.S., Lew, K., Liu, J., Pao, S.S., Paulsen, I.T.,
Tseng, T.-T., and Virk, P.S. 2000. The major facililitator superfamily. J.
Mol. Microbiol. Biotechnol. 1, 257- 280.
Stark, M.J.R. 1987. Multicopy expression vectors carrying the lac repressor
gene for regulated high-level expression of genes in Escherichia coli.
Gene 51: 255-267.
Studier, F.W., Rosenberg, A.H., Dunn, J.J. and Dubendorff, J.W. 1990. Use
Of T7 RNA-Polymerase to direct expression of cloned genes. Meth.
Enzymol. 185: 60-89.
Turner, G.J., Reusch, R., Winter-Vann, A.M., Martinez, L. and Betlach, M.C.
1999. Heterologous gene expression in a membrane-protein-specific
system. Protein Expression and Purification. 17: 312-323.
van Veen, H.W., Callaghan, R., Soceneantu, L., Sardini, A., Konings, W.N.
and Higgins, C.F. 1998. A bacterial antibiotic-resistance gene that
complements the human multidrug-resistance P-glycoprotein gene. Nature
391: 291-295.
Ward, A., Sanderson, N.M., O’Reilly, J., Rutherford, N.G., Poolman, B. and
Henderson, P.J.F. 2000. The amplified expression, identification,
purification, assay and properties of histidine-tagged bacterial membrane
transport proteins”. In ‘Membrane Transport - a Practical Approach’.
Baldwin, S.A., ed. Chapter 6, pp141-166. Blackwell’s Press: Oxford UK.
Wendt, K.U., Lenhart, A. and Schulz, G.E. 1999. The structure of the
membrane protein squalene-hopene cyclase at 2.0 angstrom resolution.
J. Mol. Biol. 286: 175-187.
Williams, K.A. 2000. Three-dimensional structure of the ion-coupled
transport protein NhaA. Nature 403: 112-115.
Williams, K.A., Geldmacher-Kaufer, U., Padan, E., Schuldiner, S. and
Kuhlbrandt, W. 1999. Projection structure of NhaA, a secondary transporter
from Escherichia coli, at 4.0A resolution. The EMBO J. 18: 3558-3563.
Yelin, R. and Schuldiner, S. 1995. The pharmacological profile of the
vesicular monoamine transporter resembles that of multidrug transporters
FEBS. Lett. 377: 201-207.
Zhang, F.M., Strand, A., Robbins, D., Cobb, M.H. and Goldsmith, E.J. 1994.
Atomic-structure of the map kinase Erk2 at 2.3-Angstrom resolution.
Nature. 367: 704-711 Feb 24.
Zhuang, J.P., Prive, G.G., Werner, G.E., Ringler, P., Kaback, H.R. and Engel,
A. 1999. Two-dimensional crystallization of Escherichia coli lactose
permease. J. Struc. Biol. 125: 63-75