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Department of Biochemistry and Biophysics, Stockholm University, Albanova University Center, SE-114 21 Stockholm, Sweden
Reprint requests to: Pär Nordlund, Department of Biochemistry and Biophysics, Stockholm University, Roslagstullsbacken 15, Albanova University Center, SE-114 21 Stockholm, Sweden; e-mail: par{at}dbb.su.se; fax: +46-8-5537-8590.
(RECEIVED October 19, 2004; FINAL REVISION November 24, 2004; ACCEPTED November 24, 2004)
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Abstract |
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Here, we present a generic and integrated parallel HTP strategy for cloning and expression screening of membrane proteins in their detergent solubilized form. Based on this strategy, we provide overall success rates for membrane protein production in Escherichia coli, as well as initial benchmarking statistics of parameters such as expression vectors, strains, and solubilizing detergents. The technologies were applied to 49 E. coli integral membrane proteins with human homologs and revealed that 71% of these proteins could be produced at sufficient levels to allow milligram amounts of protein to be relatively easily purified, which is a significantly higher success rate than anticipated. We attribute the high success rate to the quality and robustness of the methodology used, and to introducing multiple parameters such as different vectors, strains, and detergents. The presented strategy demonstrates the usefulness of HTP technologies for membrane protein production, and the feasibility of large-scale programs for elucidation of structure and function of bacterial integral membrane proteins.
Keywords: detergent screen; high-throughput; membrane proteins; multiparameter; parallel screen
Abbreviations: BL21, BL21(DE3) • C41, C41(DE3) • C43, C43(DE3) • DDM, dodecyl maltoside • E. coli, Escherichia coli • FC12, FOS-CHOLINE-12 • HTP, high-throughput
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041127005.
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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To allow various structural studies, tens or even hundreds of milligrams of highly purified protein might be needed. Thus, an efficient recombinant overexpression system for membrane proteins is in most cases a prerequisite. However, membrane proteins are not only difficult to express, but also difficult to isolate, since they require purification in detergent. Furthermore, the very hydrophobic nature of integral membrane proteins makes them hard to handle experimentally, and they can easily be "lost" upon purification, centrifugation, or gel electrophoresis. General experiences from workers in the field with the problematic experimental behavior of integral membrane proteins have lead to the expectation that these proteins are dramatically harder to produce than soluble proteins.
Presently, there are intensive ongoing efforts in development of high-throughput (HTP) expression technology for soluble protein production with success rates in the range of 50%–60% of bacterial target genes entered into the process (Kuhn et al. 2002; Lesley et al. 2002; Stewart et al. 2002; Hui and Edwards 2003). So far no HTP technologies or comprehensive success rates for membrane protein production have been reported. At present, the most extensive efforts for bacterial membrane protein production involve transporter proteins (for review, see Loll 2003). However, in order to produce sufficient amounts of such proteins for structural studies, large-scale fermentors of up to 50 L have been used (Henderson et al. 2000; Chang and Roth 2001).
An additional complication in membrane protein production is the detergent extraction procedure. The choice of detergent is a critical issue to consider, especially when designing HTP studies. There are dozens of different detergents that are commonly used, dozens more that are less characterized but still probably useful, and many novel detergents under development. Moreover, mixtures of detergents are sometimes used (Koronakis et al. 2000). It has also been reported that some compartments of the cell membrane show resistance toward certain detergents (Schuck et al. 2003). Altogether, the size of the detergent parameter space becomes very large.
Therefore, to allow larger numbers of integral membrane proteins to undergo extensive expression and purification screening, efficient parallel technology is urgently needed (see also Lundstrom 2004). To this end, we present a versatile multiparameter HTP strategy for cloning and expression screening of membrane proteins from Escherichia coli homologous to those from Homo sapiens. Furthermore, an efficient HTP detergent screen is presented. Based on these technologies, we derive initial benchmarking statistics for the production of bacterial membrane proteins in E. coli, using multiple expression vectors, strains, and conditions. We believe that the type of strategy presented is a first step toward more routine structure determination of membrane proteins, and thereby toward the generation of a wealth of information on the processes of the biomembranes, many with critical biomedical importance.
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Results |
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The two-step recombination cloning of 49 gene targets into three expression vectors using Gateway technology yielded 47 types of constructs (two cloning reactions failed), each with either a 6-His-, a FLAG-, or an MBP- at the NH2 terminus and a 6-His tag coding sequence followed by three stop codons at the COOH terminus (Table 1
). The presence of a C-terminal 6-His tag in all constructs allowed dot-blot detection of all proteins using anti-6-His probe. The constructs were successfully used to transform C41, C43, and BL21 strains.
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) and 23 proteins with N-terminal MBP-tag in C41 cells. Thus, in these cells a total of 31 distinct proteins were expressed in at least one construct (Table 2). To investigate the importance of the expression strain, the FLAG-tagged expression constructs were transformed into C41, C43, and BL21 cells. The C41 and C43 strains have been derived from BL21 to increase the yield of the membrane-targeted proteins (Miroux and Walker 1996). In total, 29 FLAG-tagged proteins were expressed: 22 in C43, 24 in C41, and 25 in BL21 (Table 2). Hence, the number of all the proteins expressed in at least one vector and strain was increased to 35 after combining all parameters (Tables 1
, 2). Based on medium-scale production experiments (see below) we estimated expression levels to be between 0.2 and 5 mg protein per liter shake flask culture.
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). These detergents either belong to families whose members have been successfully used to produce crystals, such as maltosides and glucosides (Reiss-Husson and Picot 1999), or are commonly used for protein purification in many laboratories, such as Triton and CHAPS. The amounts of detergents used during extraction and purification procedures were chosen based on the CMC for each detergent, but the detergent concentration was never <1% during the extraction. Certain detergents, such as CHAPS, may require >2% for efficient solubilization. However, we did not exceed the final detergent concentration of 2%, since that is the maximum concentration recommended for Ni-NTA agarose purification. In order to have equal starting conditions, culture growth and induction of expression were performed in shake flasks prior to transfer into 96-deep-well plates for harvest. Using the 96-well format purification described above followed by dot-blot screen, we could compare the efficiency of different detergents and identify the most suitable one for the extraction and purification of each protein (Fig. 2). Interestingly, the extraction and purification efficiency was quite the same for members of the same detergent family. Some proteins, like EM23 and EM35, could be efficiently extracted by nearly all detergents. In the case of EM43, however, FC10–FC12 were the only detergents able to solubilize high amounts of the protein (Fig. 2F). In fact, extraction and purification with FC12 always resulted in high amounts of purified proteins. Therefore, we decided to use FC12 as the standard detergent to screen for expression of all the proteins (see Fig. 1). The detergents in this series have the same head groups as phospholipids, but unlike the phospholipids they possess simple hydrophobic tails. Maltosides and thiomaltosides also resulted in high purification yield. The maltose head group is believed to increase the solubility of the members within the family as compared to the glucoside family. The maltosides are, therefore, still mild, but more efficient detergents than the glucosides.
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shows the intactness and the homogeneity of the proteins purified on gel filtration with either FC12 or DDM. The amounts of purified proteins after gel filtration were calculated to be 3–5 mg/L culture for high expression, 1–3 mg/L for medium expression, and ~0.2–1 mg/L for low expression proteins. Interestingly, the integrity of the purified proteins seems to depend on the nature of the detergent used. Some proteins tend to aggregate in the presence of FC12 (a zwittergent) but not in the presence of DDM (a neutral detergent), and vice versa (Fig. 4). Most proteins were shown to be homogenous in at least one detergent, while EM43 was the only protein that showed to be rather heterogeneous, although it is possible to separate different populations by gel filtration (Fig. 3F). EM43 is predicted to be a Co2+/Mg2+ channel based on amino acid sequence analysis. When MgCl2 was added to all the buffers during the purification EM43 was eluted as a major sharp band, corresponding to either its largest oligomeric form or nonhomogenous aggregates (data not shown). Gel filtration of EM47 resolved two major populations from each other (Fig. 3G). EM47 was not present in the void fraction and came as a separate peak, probably a tetramer. The void, however, contains an unknown protein at a higher oligomeric/aggregation form.
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Discussion |
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Our scale-up study shows a good correlation between the small-scale and large-scale experiments, suggesting that it will be possible to produce most of these proteins in milligram amounts. The behavior of these proteins during gel filtration indicates that most of them are indeed in a stable and well-folded form. Based on the gel filtration results from presented work and others (Auer et al. 2001), it is very important to choose the right detergent in order to preserve the integrity of each protein. The addition of the mini-scale His-tag purification step was beneficial for a number of reasons. First, we were able to distinguish between solubilized membrane proteins and vesicles, since the affinity of vesicles and membrane fragments to Ni-NTA agarose resin is extremely low (data not shown). Second, the expressed proteins may aggregate after extraction from the membrane, depending on the nature of the protein and the detergent used. Also, extensive delipidation has been reported to cause protein aggregation and precipitation (Auer et al. 2001; Boulter and Wang 2001; Lemieux et al. 2002). However, the precipitates cannot efficiently bind to the Ni-NTA agarose resin, and either pass through or bind to the filter. Finally, by determining the amount of purified material, a good indication of the amount of culture required for large-scale protein production needed for crystallization studies may be obtained. Standard methods for detergent screening, involving membrane preparation and ultracentrifugation, are not only time consuming, but also expensive due to the high cost of detergents. In addition, performing such a screen in a HTP manner involving a large number of detergents and proteins is not feasible. Here, we have screened 25 detergents from nine different families against 12 proteins, a total of 300 different conditions, in the course of a few hours, starting from cell lysis. Therefore, introducing the 96-well 6-His-tag purification method dramatically increases the throughput in screening solubilizing detergents of integral membrane proteins. Moreover, the method is cost effective and requires only standard laboratory equipment and handling; it can also be automated.
Since membrane proteins are idiosyncratic in their interactions with detergents, it is impossible to identify the ‘best’ detergents a priori. Ideally, a detergent (or detergent mixture) should extract and solubilize the target protein from the membrane and also have a stabilizing effect to prevent the protein from forming aggregates. In a recent report, ~20 detergents were screened in crystallization set-ups (Chang and Roth 2001). Indeed, the next rate-limiting step in membrane protein crystallography is finding the detergent suitable for crystallization screens. However, it is quite likely that the detergents that keep the protein most stable are among the best suited for crystallization. Thus, a useful subset of detergents could be identified at an early point using the presented detergent screen. Once a few detergents have been selected, other HTP approaches are needed to discard those causing larger and heterogeneous oligomers (work in progress). Thus far, we have performed large-scale purification of a dozen membrane proteins and all, except for EM43, have been successfully purified to homogeneity, of which one has resulted in diffracting crystals (data not shown).
In conclusion, the presented study demonstrates that an efficient generic HTP methodology can be implemented for the production of native bacterial integral membrane proteins. The success rate is significantly higher than anticipated, and is in fact comparable to the success rates of producing soluble bacterial proteins, although the production levels are lower. In order to implement HTP technology for structural studies on for example human membrane proteins, it is important to evaluate such technologies for the bacterial counterparts. We believe that the presented work is the first step toward this goal.
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Materials and methods |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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Recombination cloning of genes using Gateway technology
Coding sequences for the proteins on the target list were obtained from SwissProt database and used for primer design using SGD Webprimer design program.
Genes encoding proteins EM01–EM49 were amplified by PCR. Touchdown PCR was performed using Platinum Pfx DNA polymerase (Invitrogen); primers containing at least 15 gene-specific nucleotides (TAG Copenhagen, http://www.tagc.com); and the E. coli template K12
isolated with the High Pure PCR Template Preparation Kit (Roche).
Another PCR was performed using the same Pfx polymerase, primers containing attB sites (Invitrogen), and the PCR products from the touchdown reactions as templates. The products were purified using the QIAquick Purification Kit (Qiagen, VWR International).
The linear fragments flanked by attB sequences were subjected to site-specific recombination with pDONR201 vector (Invitrogen), containing the ccdB gene, flanked by attP sites and catalyzed by BP Clonase (see manufacturer’s protocols) yielding entry clones that were used to transform E. coli competent DH5
cells. Transformants were grown on LB agar (LA) plates (LabMedicine) containing 50 µg/mL kanamycin. Colonies were picked from each plate for colony PCR (using Taq polymerase and outer pDONR primers (Invitrogen)) and growth in liquid culture for subsequent plasmid preparation. Plasmid constructs were isolated using the QIAprep Spin Miniprep Kit (Qiagen).
The entry clones were subjected to another round of site-specific recombination catalyzed by the LR Clonase enzyme mix (Invitrogen) in order to subclone the genes of interest into a number of destination vectors (AstraZeneca) containing the ccdB gene flanked by attR sites, as well as the coding sequences for fusion tags (6-His, FLAG, and MBP), to generate expression clones (see Invitrogen’ protocols).
The resulting expression constructs were used to transform E. coli C41(DE3) (C41) (Avidis, Saint-Beauzire), C43(DE3) (C43) (Avidis), and BL21(DE3) (BL21) (Novagen) strains. Transformants were selected on LA plates containing 30 µg/mL tetracycline. Growth of transformants was performed as described above. Identity of all constructs was verified by dideoxy sequencing.
Protein overexpression
The cells were grown in either shake flasks or 96-deep-well plates at 37°C until the cultures reached the OD600 of ~0.8. The cultures were then cooled down to 18°C and induced overnight with 0.1 mM isopropyl-
-D-1-thiogalactoside (IPTG). Cells from 1 mL fractions were harvested by centrifugation in 96-deep-well plates and finally stored at –80°C. For gel filtration analysis, 250 mL cultures were grown and induced as described above and the cells were harvested and stored at –80°C. The typical final OD600 was between 2 and 2.5.
Cell lysis and membrane protein extraction and purification in 96-well plates
The frozen cell pellets (obtained from 1 mL of culture) in 96-deep-well plates were resuspended in 50 µL 20 mM Tris-HCl at pH 8.0, 100 mM NaCl, 1 mg/mL lysozyme, complete protease inhibitor cocktail EDTA-free (Roche), 10 U/mL benzonase (VWR International), and 1%–2% detergent according to Table 3 (extraction). Lysis and extraction were performed for 1 h at 4°C. The suspension was filtered using a 96-well 0.45 filter plate (Millipore). The filtrate was added to Ni-NTA agarose resin (Qiagen) pre-equilibrated with purification buffer (P-buffer) containing 20 mM Tris-HCl at pH 8.0, 300 mM NaCl, 5 mM -mercaptoethanol, and the appropriate detergent at a concentration above its critical micelle concentration (CMC) (Table 3). After 15 min of agitation at 4°C, the unbound material was removed by 30 sec of centrifugation at 100g. The resin was then washed with 40 mM of imidazole in the P-buffer containing the appropriate detergent at 100g for 30 sec. The bound recombinant membrane proteins were finally recovered in P-buffer containing 500 mM of imidazole and the respective detergent, by centrifugation at 100g for 1 min.
Medium-scale protein purification and gel filtration
Cell pellets from 250 mL cultures were thawed, resuspended in 20 mM Tris-HCl at pH 8.0, 100 mM NaCl, 5 mM -mercaptoethanol, and 1 mg/mL lysozyme, sonicated and centrifuged at 15,000g. The membranes were finally harvested by 1 h centrifugation at 150,000g. The membranes were resuspended and solubilized with the detergent, as stated elsewhere, in the P-buffer supplemented with 20 mM of imidazole using a glass homogenizer, followed by centrifugation for 45 min at 200,000g. The clear supernatants containing solubilized membrane proteins were loaded on Ni-NTA agarose resin pre-equilibrated with the P-buffer, including 20 mM of imidazole and the corresponding detergent. The resin was washed with the P-buffer containing 40 mM of imidazole and the corresponding detergent. The recombinant proteins were eluted with 500 mM of imidazole in the same buffer.
The integrity of purified membrane proteins was confirmed by gel filtration using Superdex 200 column (Amersham Biosciences). The yields were calculated by measuring the absorbance at 280 nm.
Dot-blot analysis
Of the sample, 1.5 µL was applied to nitrocellulose and allowed to dry. The 6-His-tagged proteins were detected using INDIA His-Probe-HRP Western blotting probe (Pierce) according to the manufacturer’s protocol. The signals were detected with FluorS-multiImager (BioRad) and quantified using the Quantity One software (BioRad).
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Footnotes |
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Acknowledgments |
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References |
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Boulter, J.M. and Wang, D.N. 2001. Purification and characterization of human erythrocyte glucose transporter in decylmaltoside detergent solution. Protein Expr. Purif. 22: 337–348.[CrossRef][Medline]
Chang, G. and Roth, C.B. 2001. Structure of MsbA from E. coli: A homolog of the multidrug resistance ATP binding cassette (ABC) transporters. Science 293: 1793–1800.
Henderson, P.J.F., Hoyle, C.K., and Ward, A. 2000. Expression, purification and properties of multidrug efflux proteins. Biochem. Soc. Trans. 28: 513–517.[Medline]
Hui, R. and Edwards, A. 2003. High-throughput protein crystallization. J. Struct. Biol. 142: 154–161.[CrossRef][Medline]
Koronakis, V., Sharff, A., Koronakis, E., Luisi, B., and Hughes, C. 2000. Crystal structure of the bacterial membrane protein TolC central to multi-drug efflux and protein export. Nature 405: 914–919.[CrossRef][Medline]
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Lemieux, M.J., Reithmeier, R., and Wang, D.N. 2002. Importance of detergent and phospholipid in the crystallization of the human erythrocyte anion-exchanger membrane domain. J. Struct. Biol. 137: 322–332.[CrossRef][Medline]
Lesley, S.A., Kuhn, P., Godzik, A., Deacon, A.M., Mathews, I., Kreusch, A., Spraggon, G., Klock, H.E., McMullan, D., Shin, T., et al. 2002. Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline. Proc. Natl. Acad. Sci. 99: 11664–11669.
Loll, P.J. 2003. Membrane protein structural biology: The high throughput challenge. J. Struct. Biol. 142: 144–153.[CrossRef][Medline]
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Miroux, B. and Walker, J.E. 1996. Over-production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and gobular proteins at high levels. J. Mol. Biol. 260: 289–298.[CrossRef][Medline]
Reiss-Husson, F. and Picot, D. 1999. Crystallization of membrane proteins. In Crystallization of nucleic acids and proteins (eds. Ducruix, A. and Giegé, R.), pp. 245–268. Oxford University Press, New York, NY.
Schuck, S., Honsho, M., Ekroos, K., Shevchenko, A., and Simons, K. 2003. Resistance of cell membranes to different detergents. Proc. Nat. Acad. Sci. 100: 5795–5800.
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