JAC Advance Access published online on April 5, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm036
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A genomic strategy for cloning, expressing and purifying efflux proteins of the major facilitator superfamily
1 Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, Institute for Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, UK 2 Division of Biochemistry and Molecular Biology, Glasgow Biomedical Research Centre, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8TA, Scotland, UK 3 Department of Bacteriology and Immunology, Veterinary and Agrochemical Research Centre, VAR-CODA-CERVA, Groeselenberg 99, B-1180 Ukkel, Belgium 4 Department of Pharmaceutical Biosciences, University of Oslo, PO BOX 1068 Blindern, 0316 Oslo, Norway
* Corresponding author. Tel: +44-113-343-3175; Fax: +44-113-1407; E-mail: p.j.f.henderson{at}leeds.ac.uk
Received 12 July 2006; returned 3 August 2006; revised 23 January 2007; accepted 26 January 2007
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A genomic strategy for the overexpression of bacterial multidrug and antibiotic resistance membrane efflux proteins in Escherichia coli is described. Expression is amplified so that the encoded proteins from a range of Gram-positive and Gram-negative bacteria comprise 5% to 35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGS(His)6-tag or a Strep-tag at the C terminus. These tags facilitate the purification of the overexpressed proteins in milligram quantities for structural studies. The strategy is illustrated for the bicyclomycin resistance efflux protein, Bcr, of E. coli.
Key Words: membrane transport proteins , antibiotic resistance , bicyclomycin , multidrugs , pathogens , Helicobacter pylori , Brucella spp. , Escherichia coli
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The phospholipid inner cell membrane of bacteria is inherently impermeable to many nutrients and waste products of metabolism. The uptake of nutrients into the cell and the secretion of wastes out of the cell, therefore, depend on the presence of transport proteins. These transport proteins comprise 3% to 15% of genomic potential in all organisms, and the determination of their structures is the major bottleneck in the quest to understand the detailed molecular mechanisms of membrane transport. Their activities are usually energy-linked in bacteria, and this allows transport against the prevailing electrochemical gradient of the solute.1 In the case of drugs or antibiotics, these molecules often penetrate the cell membrane and are then actively secreted from the cell, thereby conferring resistance. The low abundance of these membrane proteins and their hydrophobic nature make them difficult to isolate in amounts required for the elucidation of their three-dimensional (3-D) structures.
Analyses of the available bacterial genomes predict that membrane transport proteins likely to catalyse drug efflux comprise 2% to 7% of the protein complement. This is in contrast to their much lower percentage of expressed cell protein.2,3 Like the majority of membrane transporters, drug efflux proteins fall predominantly into the two classes1–3 of the ATP-binding-cassette (ABC) superfamily4,5 or the major facilitator superfamily (MFS),5–9 which is usually energized by the electrochemical gradient of protons (sometimes sodium ions) in an antiport mechanism. In addition, there are at least three more classes of efflux transport systems.2,3 The ‘small multidrug resistance’ family, itself a subfamily of the drug metabolite transporter (DMT) family of transporters;10,11 the ‘resistance/nodulation/division’ (RND) family2,10,12 members of which, e.g. the AcrABTolC complex,13,14 are especially important for antibiotic resistance of Gram-negative pathogens; and the ‘multi-antimicrobial extrusion’ family (MATE), a subfamily of the ‘multidrug/oligosaccharidyl-lipid/polysaccharide’ (MOP) flippase superfamily.2,15 The distribution of each of these types of efflux systems varies considerably in different microorganisms.2,3 For example, in Escherichia coli, the ratio of predicted ABC:MFS:RND:MATE:DMT is 7:32:9:4:6, whereas in Bacillus subtilis, it is 4:32:1:4:7.3
Although the actual substrates of many efflux systems have yet to be identified, there is a growing awareness that they can contribute to drug and antibiotic resistance in infectious microbes and that we should be actively seeking new drugs that inhibit their activities.2,10–12,16 Many individual efflux proteins have a wide range of substrates and contribute to ‘multidrug resistance’.11–16
In this article, a strategy is described that enables the amplified expression and purification of bacterial membrane transport proteins, from several species of bacteria, in amounts required for structural studies. Many of these prokaryote transport proteins are homologous to members of the MFS found in a range of higher organisms, such as protozoan parasites, fungi, plants and mammals.2,3,6,7,9,17,18 It is, therefore clear that results of structure–activity studies of these bacterial proteins will be relevant to the understanding of the corresponding eukaryotic transporters. In other cases, the transport systems are unique to bacteria and, indeed, sometimes critical for growth in cases where these bacteria are pathogenic. The availability of purified active protein, of these key transporters, may then be useful for the discovery of novel antibacterials. This article focuses on efflux proteins of the MFS, which dominate in many bacteria.2,3 For example, as mentioned earlier in E. coli K12, there are 18 MFS, 7 ABC, 6 RND, 4 DMT and 2 MOP efflux proteins. In the post-genome age, identification of possible efflux proteins through similarities of their amino acid sequences is trivial,8,17,18 but their complete biochemical, functional and structural characterization in purified systems is only just beginning. Thus, our focus is on structural genomics—devising a general strategy to move quickly from identification of a gene of interest to having sufficient quantities of pure protein for functional and structural studies.
Our approach is illustrated by the overexpression and purification of the bicyclomycin resistance (Bcr) efflux protein of E. coli.19,20 The activity of the Bcr protein was characterized previously19 and the similarities of its amino acid sequence to many other efflux transporters were noted. Bcr belongs to the MFS of transport proteins and its hydropathy profile suggests that it has 12 transmembrane 
-helices.19,20
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Introduction of the gene encoding a putative efflux protein into the plasmid pTTQ18
The genes encoding the efflux proteins were amplified from the corresponding samples of genomic DNA using appropriate mutagenic oligonucleotides. These oligonucleotides were designed to introduce an EcoRI site at the 5' end and a PstI site at the 3' end, in order to promote the subsequent ligation with the 4.56 kb pTTQ18/RGS(His)6 fragment. The resulting PCR product was isolated from an agarose gel and then digested with EcoRI and PstI.
In order to clone each of the genes into the pTTQ18 plasmid vector,21–25 the plasmid pNorAH6 (pTTQ18 containing the gene norAH6) was isolated from E. coli strain BLR and digested with the restriction endonucleases EcoRI and PstI to yield two DNA fragments of 4.56 and 1.2 kb. The larger fragment [pTTQ18 with the RGS(His)6 coding DNA sequence] was isolated from an agarose gel.
Ligation reactions were performed using the EcoRI–PstI digested gene and pTTQ18–RGS(His)6 fragments at various vector:insert molar ratios. The ligated product was subsequently transformed into E. coli XL1 Blue StratageneTM {recA1, endAI, gyrA96, thi-I, hsdR17, supE44, relA1, lac [F'proAB lacIq Z
M15, Tn10 (TetR)]} or XL10 Gold StratageneTM {recA1, endAI, gyrA96, relA1, lac [F'proAB lacIq ZM15, Tn10 (TetR) Amp Cam]} cells and recombinant clones were selected on Luria broth (LB) plates containing carbenicillin. Automated DNA sequencing was used to confirm the presence of each gene and the absence of any adventitious base changes. The plasmid was then transformed into E. coli strain BL21 (DE3) NovagenTM [F– ompT hsdSB(rB– mB–) gal dcm (DE3)] for expression studies.
This procedure can be applied to any gene that does not include EcoRI or PstI restriction sites. If these sites are present in the coding region, then EcoRI and PstI can still be introduced as flanking sites with partial digestion used to obtain a fragment uncut at the internal site(s), or a two-step procedure adopted (see, for example, Potter et al.26). Alternatively, other flanking restriction sites compatible with the multicloning site in pTTQ18 can be chosen,21 for example, BamHI at the 5' and HindIII at the 3' end of the gene.
If the C terminus of the target membrane protein is predicted to lie in the periplasm, then a Strep-tag may well be successful if the RGS(His)6-tag is not.
Expression of the target protein and isolation of the membrane
For small-scale investigation of protein expression, 50 mL of cultures in 250 mL flasks were used. Maintenance and growth of these E. coli BL21 strains were achieved by culturing the bacteria in either LB liquid medium, minimal salts medium containing 20 mM glycerol (M9), double strength yeast extract-tryptone (2YT) or on plates of the before-mentioned medium containing 1.5% agar. Carbenicillin (at least 100 mg/L) was used throughout all stages of growth, in order to maintain plasmid integrity.
Total membranes were prepared from sphaeroplasts by the water lysis method.24 Inner membrane vesicles were prepared from 500 mL of cultures in 2 L baffled conical flasks or from 30 L of fermentor cultures.27 After harvesting, the cells were disrupted by explosive decompression using a Constant Cell Disruption System (www.constantsystems.com). The inner and outer cell membranes were separated by sucrose density centrifugation, followed by washing in buffer to remove the sucrose and EDTA.8,24
For both small-scale tests and larger scale production of inner membranes, the growth of the E. coli strains was allowed to continue until the cell density had reached an A680 of approximately 0.6. At this point, the expression of the cloned gene was induced by the addition of isopropyl-ß-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. Growth sometimes continues for 3–4 h following the induction of the tac promoter. However, for many of the cloned efflux proteins, the induction of expression attenuates growth and in extreme cases, the cells may even lyse. Accordingly, it is important to undertake pilot experiments to optimize cell and protein production, e.g. in different media with different concentrations of IPTG, using different periods of induction, and possibly different temperatures for growth.
Detection of expressed histidine-tagged transport proteins in E. coli membrane preparations
E. coli BL21(DE3) cells harbouring each plasmid were cultured in 2YT medium and expression trials performed with different concentrations of IPTG (0.0–1.0 mM). IPTG at a concentration of 0.5 mM was sufficient for the maximal expression of the putative Bcr(His)6 protein.
Membrane samples23,24 were prepared for analysis of the overexpressed protein by SDS–PAGE. An IPTG-inducible protein was observed to migrate at a molecular mass of 
33 kDa for Bcr(His)6 (Figure 1). It is common for membrane proteins to migrate at 65% to 75% of their true molecular weight, possibly as a result of their hydrophobicity, high binding of SDS or the retention of secondary/tertiary structure accelerating their passage through the gel.24 For example, the predicted molecular weight of Bcr(His)6 is 45 169 Da, compared with the apparent mass of 31–33 kDa from SDS–PAGE (above and Figure 2). Scanning densitometry analyses of the gels showed that the induced protein was expressed at 12% to 16% of total membrane proteins, indicating that overexpression has occurred. In comparison, the protein at the same position in the uninduced sample is only expressed at 3% of the inner membrane protein. The identity of the overexpressed protein in the membranes was confirmed by western blotting (Figure 1), using an antibody to the C-terminal RGS(His)6-tag.23–25
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Solubilization and purification of histidine-tagged transport proteins
Initial purification of Bcr(His)6 was achieved by immobilized metal affinity chromatography (IMAC,22 Figure 2). The identity of the protein was confirmed by western blotting, and by N-terminal sequencing. Some minor contaminants are visible and bands of higher molecular weight may be oligomers of the protein (Figure 2).
It is important to have rigorous tests for both the identity and the integrity of the purified overexpressed proteins before further, often time-consuming, studies are undertaken.
The conditions for solubilization and purification, i.e. dodecyl-ß-D-maltoside (DDM) concentration for solubilization and imidazole concentration for IMAC, vary depending on the characteristics of each individual transport protein. However, the generic conditions described here have proved generally useful. The efflux transporters were initially solubilized in 20 mM Tris pH 8.0, 20 mM imidazole pH 8.0, 300 mM NaCl, 20% glycerol and 1% DDM. After washing out the non-specifically bound proteins, Bcr(His)6 was eluted by the following buffer: 20 mM Tris pH 8.0, 200 mM imidazole pH 8.0, 150 mM NaCl, 5% glycerol and 0.05% DDM.
Confirmation of integrity and monodispersity of the purified protein
Before crystallization trials are undertaken, it is important not only to confirm the integrity of the overexpressed protein but also to test for its monodispersity. Monodispersity is important because aggregated material usually inhibits crystallization. Size exclusion chromatography is an established technique for the purification of proteins on the basis of their size. In the case of membrane proteins isolated in detergents, the apparent molecular size is increased by the presence of the detergent micelle.28 Even so, this method can be used as both a purification step, in addition to IMAC, and a way to assess monodispersity.
Measuring the spectrum of absorbances of circularly polarized light [circular dichroism (CD)] in the far UV/UV range of wavelengths is a useful technique for the detection of secondary structure within proteins in solution and can be used as an indicator, for membrane proteins, of -helical content.29 For example, the CD spectrum obtained for the purified (His)6-tagged Bcr(His)6 protein (Figure 3) reveals a high -helix content. This is a convenient test in a protein chemistry laboratory for confirming the retention of integrity during purification.
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Importantly, the activity of the isolated protein still requires confirmation. This can be achieved if there is a high affinity substrate or inhibitor suitable for direct binding studies, or by reconstitution into artificial membranes, where the transport activity of the protein can be measured.30
Crystallization trials on the purified proteins
Following purification and checks for structural integrity, crystallization trials were set up ( ± imidazole, ± glycerol) using sparse-matrix screens from Hampton, Sigma and Molecular Dimensions. At present, we prefer to retain glycerol in the crystallization drop as this provides a readymade cryo-solution if/when crystals appear. Crystallization trials are also underway with the addition of reserpine to the crystallization drop. Reserpine is a general inhibitor of MFS efflux systems,16 and it is hoped that this compound will lock the protein into a single conformation so as to facilitate the formation of crystals. However, hydropathy plots of many efflux protein sequences reveal very few hydrophilic regions between the transmembrane regions, and it is generally thought that these hydrophilic regions are required for type II crystal formation. Therefore, crystallization trials have also been performed on the basis of the lipidic cubic phase technique,31 using the dedicated screen developed by Emerald Biosystems. These trials are also ongoing.
Two-dimensional crystals suitable for electron diffraction crystallography were successfully prepared for the overexpressed Tet(A) MFS tetracycline transport protein from E. coli,32 but their level of resolution was insufficient for structure determination.
Recently, a model has been proposed for the E. coli EmrD efflux protein based on X-ray diffraction data.33 Previously, such models were derived from tenuous relationships to more distantly related sugar transport proteins.34 The EmrD protein model shows the expected 12-helix structure with an interior composed mostly of hydrophobic residues, consistent with its role in transporting amphipathic molecules.33
Wider application of this strategy for the amplified expression and purification of membrane proteins
The same strategy, with minor modifications in growth and purification conditions, has been used for overexpression of other membrane transport proteins from E. coli, B. subtilis, Helicobacter pylori and other bacteria, both Gram-negative and Gram-positive.3,22,23 Most attempts have so far been successful, i.e. the induced cloned protein comprised at least 10% of the inner membrane of the E. coli host strain, and in every case where the (His)6-tag was added at the C terminus, the protein has been purified successfully by IMAC.
For each protein, the growth conditions need to be optimized in 1–25 L cultures of E. coli host strains, testing both minimal and complex media at temperatures between 25°C and 37°C in order to maximize expression.24 The concentration of IPTG required is routinely tested between 0.0 and 1.0 mM, 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). Similarly, the period of exposure to IPTG (2–24 h) is investigated in order to obtain maximum expression. In many cases, a 25 L fermentor can conveniently be used without compromising expression, although for some proteins, the level of expression is always higher in batch cultures of 500–800 mL in 2 L baffled flasks. Further examination of the parameters regulating growth and protein production in these conditions may enhance our understanding of expression and enable us to reproduce the complex growth behaviour of flask culture in the more controlled environment of a fermentor.
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The prime purpose of this article is to illustrate a generic procedure (Figure 4) for obtaining sufficient quantities of correctly folded efflux protein(s) from a variety of microorganisms, including pathogens, for structural studies. We have achieved this for many such proteins from both Gram-positive and Gram-negative organisms such as B. subtilis, Brucella melitensis, Campylobacter jejuni, E. coli, H. pylori, Neisseria meningitidis, Streptomyces coelicolor and Staphylococcus aureus.
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Target selection can follow at least two precepts. A single efflux protein may be selected for investigation because its activity is thought to be of importance to the infectivity of an established pathogen. Alternatively, in order to reduce the risk of failure in determining a 3-D structure, a series of orthologues can be selected from the same and different organisms. These orthologues should be related by amino acid sequence (at least 20% identity in aligned sequences), so that their 3-D structures can be assumed to be homologous. In the second case, a broad-based trial of amplified expression, purification, and stability is then conducted on all targets in parallel. At each stage, it is to be expected that some will prove unsuitable, so by a process of elimination, starting from about 40 candidates, one hopes to arrive at a panel of 5–10 proteins that may crystallize. Of these, there may be one to two that yield crystals that diffract X-rays to sufficient resolution to allow structure determination.
It is possible to proceed from identification of a gene that encodes a membrane protein in a bacterial genome to the production of milligram quantities of purified protein in a few weeks, and the application of high throughput methods for cloning and purification will hopefully reduce this time still further.35 The yield and purity of protein may well be increased by further optimization of conditions, especially for detergent extraction of protein from the membrane. Our typical yields are sufficient to initiate 3-D crystallization trials for X-ray crystallography and nuclear magnetic resonance (NMR) studies.36,37
In addition, the purified protein can be examined by a variety of biophysical techniques: mass spectrometry for precise Mr and sequence determination; CD and Fourier transform infrared spectroscopy to measure the content of secondary protein structure; fluorimetry and calorimetry to measure the ligand binding and electron spin resonance spectroscopy to investigate the protein–lipid interactions. These can also illuminate structure–activity relationships, especially when performed in conjunction with site-directed mutagenesis and genetic recombination. Expression of genes encoded in the pTTQ18 vector is independent of the E. coli host in our experience so far, and movement between host strains by transformation is easily accomplished in order to optimize, for example, specific labelling with stable isotopes for NMR studies.
In recent landmark articles, the 3-D structures of the lactose (LacY)38 and -glycerophosphate (GlpT)39 and possibly the EmrD (efflux)33 MFS transport proteins of E. coli have been determined by X-ray crystallography. These structures can now be used to make better models of the structures of related proteins,40 including those responsible for efflux.35 The molecular origin of the promiscuity of many ‘multidrug’ substrates is consequently becoming apparent.33,41 Future elucidation of the structures of efflux proteins from many pathogenic microorganisms may very well uncover new targets for preventing or treating bacterial infections, an increasingly urgent requirement as ‘superbugs’ acquire apparently unconquerable resistances.11,12,16,35,36
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None to declare.
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This work was funded by the European Membrane Protein consortium (EMeP, contract LSGH-CT-2004-504601), the EU COST Action B16, BBSRC, and by equipment grants from the Wellcome Trust and BBSRC. M. S. is grateful to the Iranian Government for financial support. MRC provided a studentship for K. E. B. We thank Dr J. G. Keen for N-terminal amino acid sequencing of the purified proteins. We also acknowledge the prior studies of Dr S. L. Palmer and the constructive interest of Dr M. Gwynne (GSK), Dr F. Paul (GSK) and Dr G. Badman (GSK). P. J. F. H. is grateful for a Leverhulme Research Fellowship.
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