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Membrane protein expression: no cells required
Review
Membrane protein expression: no cells required Federico Katzen, Todd C. Peterson and Wieslaw Kudlicki Life Technologies, 5791 Van Allen Way, Carlsbad, CA 92008, USA
Structural and functional studies of membrane proteins have been severely hampered by difficulties in producing sufficient quantities of properly folded protein products. It is well established that cell-based expression of membrane proteins is generally problematic and frequently results in low yield, cell toxicity, protein aggregation and misfolding. Owing to its inherent open nature, cell-free protein expression has become a highly promising tool for the fast and efficient production of these difficult-toexpress proteins. Here we review the most recent advances in this field, underscoring the potentials and weaknesses of the newly developed approaches and place specific emphasis on the use of nanolipoprotein particles (NLPs or nanodiscs). Introduction Despite the extraordinary differences among organisms belonging to each of the three domains of life, nearly a third of their gene products, in all cases, are destined to be inserted into a lipid bilayer. These products often form supramolecular complexes that operate in diverse and often essential ways. Examples of their biological activities include, but are not limited to, energy generation and conversion in the respiratory chain, cell division, signal transduction and transport processes. Perturbation of their biological activities is one of the primary responses of the cell against infections or in genetic disorders such as cancer. These and other reasons have put membrane proteins in the spotlight of the drug development industry, a statement that is reflected by the fact that they account for over 50% of the current pharmaceutical targets [1]. The natural abundance of membrane proteins is usually too low to purify sufficient material for functional and structural studies. Furthermore, in vivo overexpression of these types of proteins is notoriously problematic, often resulting in low yield, cell toxicity, protein aggregation and misfolding. These hindrances are frequently the consequence of processes that affect cell viability, such as membrane overcrowding or the shortage of the insertion machinery (i.e. the SEC translocon, see Glossary) required for the expression of essential proteins. Another difficulty is that membrane proteins are naturally embedded in a complex and dynamic lipid bilayer, which limits the use of many standard biophysical techniques and further complicates their purification and handling. For example, reconstituting purified membrane proteins into a bilayer support is not a trivial endeavor [2,3]. For a fairly complete picture of the challenges that Corresponding author: Kudlicki, W. ([email protected])
the study of membrane proteins poses, we recommend consulting a seminal review in this area [4]. Cell-free protein expression systems are increasingly being considered as viable alternatives for overcoming the above obstacles, as cell viability and protein expression processes are virtually decoupled in these systems (for indepth reviews on cell-free expression technologies, readers should consult Refs [5–12]). In addition, the open nature of the system allows direct access to the reaction conditions allowing, for example, the supply of accessory reagents, such as lipids (in various forms), folding catalysts or a variety of amphiphilic molecules (Figure 1 and see below). The expansion this field has experienced over the past decade has resulted in significant improvements in the yield, solubility and quality of the membrane proteins. For example, progress made in this area has recently led to the publication of the first crystal structure of a membrane protein produced in vitro [13]. Here, we almost exclusively discuss the most recent advances in this field. For earlier accomplishments, we encourage the reader to consult previous reviews [14–17]. This manuscript primarily focuses on the crucial aspects of the reaction that need to be considered to maximize yield and to facilitate downstream applications of membrane proteins expressed in vitro. Cell-free protein expression in the presence of detergents Perhaps the most obvious strategy for facilitating the expression of membrane proteins in vitro is through the Glossary Apolipoprotein: any of various proteins that combine with a lipid to form a lipoprotein and are a constituent of cholesterol-carrying molecules. Asymmetric droplet interface bilayer: membrane formed by joining lipidmonolayer-encased aqueous droplets that are submerged in an oil–lipid mixture. Critical micellar concentration: concentration of surfactants above which micelles are spontaneously formed. Inclusion body: nuclear or cytoplasmic aggregate of stainable substances, usually proteins. Liposome: a closed lipid vesicle surrounding an aqueous interior. Nanolipoprotein particle (or nanodisc): a discoidal particle of defined and controllable size formed by a phospholipid bilayer planar segment surrounded by a protein coat (scaffold protein). Phase transition: the transformation of a thermodynamic system from one phase to another. At the phase-transition point, physical properties may undergo abrupt change. Proteoliposome: a liposome into which one or more proteins have been inserted, usually by artificial means. Synthetic biology: an area of biological research that combines science and engineering in order to design and build (‘synthesize’) novel biological functions and systems. Translocon: protein complex associated with the translocation of nascent polypeptides across membranes.
0167-7799/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2009.05.005
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Figure 1. Current strategies for the cell-free expression of membrane proteins. The figure shows the key reagents and products involved in each of the approaches further described in the text. For simplicity, reactants intrinsic to the extract as well as buffer are omitted. The gene encoding for the membrane protein is present in all strategies, whereas the associated reagents, such as detergents, liposomes, NLPs or the scaffold-encoding gene, vary depending on the particular approach. (a) Reaction in the presence of detergents. If no additional reagents are added, the protein will be associated with detergent micelles. Alternatively, proteoliposomes can be reconstituted from the detergent-protein micelles, as indicated in the text. (b) Expression of membrane proteins as a precipitate. Proteoliposomes can be reconstituted by solubilizing the protein precipitate with detergent and liposomes. Alternatively, the protein might be solubilized in detergent micelles upon the addition of a mild detergent. (c) Expression in the presence of liposomes or vesicles. Here, liposomes are added directly to the reaction. (d) Expression in the presence of pre-fabricated NLPs. (e) Co-expression with scaffold proteins. Both target and scaffold proteins are expressed in vitro and protein–NLP complexes are then formed in situ. Components shown are not drawn to scale.
addition of detergents to the reaction vessel (Figure 1a). Several publications have addressed the effect of detergents on the productivity, solubility and activity of membrane proteins expressed in vitro. Although the final outcome strongly depends on the protein being expressed, a few commonalities are apparent from the published literature (for comprehensive lists, see Refs [14,18]). Successful experiments in terms of yield and solubilization were those that used mild detergents with relatively low critical micellar concentration (CMC), such as polyoxyethylene-alkyl-ethers (e.g. Brij 35, Brij 58 and Brij 78), digitonin, 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) and n-dodecyl-b-D-maltoside (DDM) [19–22]. As a general rule, to prevent the formation of aggregates or inhomogeneous proteomicelles, the molar ratio between the expressed protein and the micelles must never be higher than 1.0. Detergents with relatively high CMC are generally not recommended because they must be present in rather high concentrations, therefore inhibiting transcription and translation [18]. However, a few exceptions to this rule have been reported, as in the case of torque-generating proteins (such as Vibrio alginolyticus PomA and PomB), which were expressed in the presence of the detergent CHAPS [23]. Furthermore, small proteins such as efflux transporters have been demonstrated to be more tolerant to a wider range of detergents, including polyethylene glycol derivatives such as Triton X-100 [24] or fluorinated surfactants [25]. 456
The major shortcoming of the use of detergents to solubilize membrane proteins is the uncertainty of how well micelle structures mimic the natural membrane protein environment. For example, the absence of a bilayer might have adverse consequences on the structure and activity of proteins [4]. In addition, detergents are denaturants per se, making it difficult to study protein macromolecular structure and function. Overall, detergents provide a convenient means of solubilizing and handling membrane proteins during in vitro synthesis. However, a method of maintaining membrane proteins in a native-like bilayer would greatly extend the accuracy and facility of membrane protein enzymology. Reconstitution or direct insertion into vesicles and liposomes An ideal situation for in vitro membrane protein reconstitution would be to work in an environment that more closely resembles the natural lipid bilayer. A few strategies are being developed to achieve this aim. One of them relies on the incorporation of the membrane protein into liposomes (Figure 1a,b). This approach requires, as a starting point, that the membrane protein has been purified and solubilized (not necessarily in its active form) in detergent micelles. The virtual absence of hydrophobic compartments in a cell-free environment promotes precipitation of hydrophobic proteins, which, in contrast to proteins present in inclusion bodies, can be quickly and effectively
Review solubilized in mild detergents (Figure 1b) [14,26]. In particular, 1-myristoyl-2-hydroxy-sn-glycero-3-[phosphorac-(1-glycerol)] (LMPG) was found to be extremely efficient in solubilizing this type of precipitate [18,27]. Therefore, making membrane proteins insoluble in vitro will facilitate their purification and might constitute a suitable starting point for their reconstitution into liposomes. However, reconstitution experiments must take place under non-equilibrium conditions and so are difficult to stage. Furthermore, no single strategy was shown to serve equally well for all membrane proteins [28] and, consequently, the protocol has to be adjusted for each protein studied. Reconstitution examples of membrane proteins expressed in vitro include those based on the freeze–thaw technique [26,27,29] or using detergent adsorbent particles in the presence of liposomes [30,31]. The freeze–thaw technique is based on the demonstration that freeze–thawing of liposomes mixed with protein enhances the formation of proteoliposomes. Liposomes can be made from a variety of lipids, such as phosphatidylserine, phosphatidylcholine, cholesterol, E. coli lipids or asolectin. Another strategy is to perform the cell-free reaction in the presence of unilamellar liposomes (Figure 1c). Although the mechanism by which proteins are inserted into naked membranes remains elusive, it is apparent that the insertion efficiency is strongly dependent on the phase transition temperature, the length of the lipid and the overall hydrophobic thickness of the bilayer [32]. Successful expression of a variety of functional proteins, such as stearoyl-CoA desaturase, bacteriorhodopsin, apoCytochrome b5 and the catalytic core of the NADPH oxidase complex, has been reported using liposomes made of a variety of lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), phosphatidylcholine, soybean lipids and spinach thylacoids [32–36]. Some of these functional proteoliposomes have been identified as good candidates for the delivery of therapeutic proteins [35,36]. A disadvantage of working with unilamellar liposomes is the relative heterogeneity of the samples and the presence of residual multilamellar vesicles [37]. This feature prevents the application of this strategy in structural biology studies. For example, liposome particles ranging widely in size between 30 and 200 nm are commonly obtained using currently established protocols [15]. Although procedures for preparing more homogeneous liposome populations exist [38], these require unusual equipment and are not widely used. Another approach stems from the rationale that most of the membrane proteins in Gram-negative bacteria are targeted to their final location by processes that depend on the signal recognition particle (SRP) receptor, the SecYEG translocon and YidC, which are components embedded in the natural target membranes [39]. These components can be added to the reaction as part of inverted vesicles made from E. coli cells (Figure 1c), as proposed by Kuruma and coworkers [40]. These authors reconstituted the E. coli membrane integration and translocation mechanisms using a defined cell-free system that was supplemented with E. coli inverted vesicles and revealed that no cytosolic factors other than the SRP and SecA are required
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for membrane protein integration [40]. More recently, Wuu and Swartz reported the high-yield expression of functional integral membrane proteins using an energy-efficient cell-free system supplemented with E. coli inner membrane vesicles [41]. Although this approach addresses the expression of Sec- and SRP-dependent proteins, it relies on the availability of high-quality inverted E. coli vesicle samples, which are difficult to prepare and, to our knowledge, not commercially available. By contrast, crude vesicles derived from dog pancreas have been commercialized for over two decades; however, they have a profound negative effect on the translational rate of the resulting lysate [42]. Finally, the relatively high heterogeneity of vesicle preparations in general might preclude using reaction products embedded in these structures for structural studies. This drawback is resolved by new technologies, as discussed below. Use of nanolipoprotein particles To overcome the shortcomings stemming from the use of microsomes and vesicles, novel strategies based on the use of ‘nanodiscs’ or ‘nanolipoprotein particles’ (NLPs) have been recently proposed (Figure 1d,e). NLPs are nanometersized, discoidal particles comprising amphipathic helical proteins (scaffold proteins) that wrap themselves around the planar circumference of a lipid bilayer (for a fairly complete review on NLPs, see Ref. [37]) (Figure 2a). A variety of scaffold proteins, such as apolipoproteins A1, E and C and insect lipophorins, have been shown to spontaneously self-assemble into NLPs in the presence of phospholipids [43,44]. Contrary to what is observed with liposomes or vesicles, NLP preparations are remarkably monodisperse (molecular mass deviation <5%) owing to the scaffold protein being the major determinant of their average diameter (9 to 20 nm depending on the type of scaffold) [43–45]. Other advantages of NLPs compared with liposomes include: (i) better representation of the phase transition behavior of biological membranes [37]; (ii) simplicity of preparation (no unconventional instrumentation required); and (iii) unrestricted access to both faces of the lipid bilayer. One of the cell-free NLP approaches for producing membrane proteins makes use of preformed (empty) NLPs that are added to the cell-free reaction and thus serve as a membrane support for the membrane protein [46,47] (Figure 1d). The products of the reaction are NLP–protein complexes, empty NLPs and residual precipitated membrane proteins. Virtually pure soluble complexes can be readily purified by means of a tag fused to the membrane protein (precipitated material is removed by a short precentrifugation step) or, even more stringently, by tandem purification using a second peptide tag fused to the scaffold protein. A comprehensive expression study of many biologically important membrane proteins of different topologies, origin, sizes and proposed roles showed that, without exception, all tested proteins dramatically increased their solubility in the presence of NLPs [46,47] (Figure 2c). Importantly, analytical assays for a subset of these model membrane proteins indicated that the proteins were also correctly folded and active (for an example, see Figure 2b). The approach has been validated using cell-free extracts 457
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Figure 2. Cell-free protein expression in the presence of NLPs. (a) Illustration of a complex between an NLP and a membrane protein. Transmembrane segments (a helices) are shown in red, and the outer gray shell represents the scaffold protein ring. (b) Activity of the E. coli multidrug transporter EmrE expressed in vitro. Radioactive tetraphenylphosphonium (TPP+)-binding was determined. Binding constants are in agreement with previous determinations [46]. As shown in the inset, [3H]TPP+ binding was performed either in the absence (empty bars) or presence (filled bars) of nonradioactive TPP+. Binding reactions were carried out with EmrE synthesized in the presence (+) or absence ( ) of NLPs, demonstrating that enzymatic activity was only obtained in the presence of NLPs. Adapted from [46]. (c) Solubility of a set of proteins expressed either in the presence (filled bars) or absence (empty bars) of NLPs. In the presence of NLPs, protein solubility was significantly increased. For the analyzed dataset, the overall solubility increased from 17.3 2.2% to 78.8 3.4%. GenBank accession numbers and number of predicted transmembrane segments (TMSs) of their protein products are indicated. Black, red and green annotations identify the gene source as human, murine and bacterial, respectively [46]. Reproduced from [46] with the permission of ACS Publications.
derived from wheat germ, rabbit reticulocytes and E. coli cells [46]; however, only the latter has been consolidated into a commercially available kit [48] (MembraneMaxTM Protein Expression Kit, Life Technologies). Another advantage of the system is that, as opposed to the above-mentioned pancreatic dog microsomes, NLPs exhibit no detrimental effects on the translation efficiency of the lysates [46]. Yield is protein dependent and linearly scalable, topping at 0.9 mg/ml of soluble product in the case of bacteriorhodopsin [46]. The main limitation to this approach, as with the liposomes, relates to the absence of a translocon (and also a transmembrane potential), which is required for the proper insertion and folding of a variety of integral membrane proteins [39,49]. However, reconstitution of the bacterial SecYEG complex into NLPs has been successfully attained [50,51], setting the scene for an assisted-insertion mechanism of Sec-dependent proteins into the particles. Finally, it is worth mentioning that the NLP–protein association process in the absence of a translocon is believed to be cotranslational, as no complexes are formed after blocking the peptide chain 458
elongation activity of the extract [48]. To insert a pre-made protein into the NLPs, a reconstitution approach should be followed [44]. A second cell-free NLP strategy stems from the ability of apolipoproteins (fat-bound proteins) to sequester lipid bilayer patches in solution. In this approach, the scaffold protein is co-expressed with the target membrane protein in the presence of liposomes, resulting in the in situ assembly of empty and target-filled NLP structures [52] (Figure 1e). The benefit of this method is its inherent simplicity, as there is no need to produce NLPs before the assay. However, phospholipids need to be processed in advance to obtain suitable unilamellar liposomes. The drawback of this method is that the NLP–protein complex has to be purified out of a large set of reaction products consisting of liposomes, proteoliposomes and lipoprotein particles of varying sizes. In addition, the ratio between the two programming DNA templates (coding for the scaffold and target proteins) has to be adjusted for each individual protein target. This approach has been validated for batch and continuous exchange formatted reactions [52]. Yields
Review are considerably lower than when using pre-assembled NLPs, as an important fraction of the resources has to be dedicated to the expression of the scaffold protein. The methods described above highlight the need to distinguish particles that can differ by as little as a fraction of a nanometer (in height). For that purpose, atomic force microscopy has proven to be a very robust technique, allowing the rapid scanning of a surface containing hundreds of particles and reliably discriminating between empty and filled NLPs [45,53]. Future perspectives Although cell-free protein expression has already proven to be an extraordinary vehicle for studying membrane proteins, it still faces major challenges. For example, an area within this technology that still warrants further improvement is structural biology. Although some structures have been resolved using X-ray crystallography and solid state NMR [13,54], a successful outcome is not the typical case (for a review see Ref. [54]). It would be interesting, for instance, to examine the synergies between the cell-free-NLP approach and crystallization in lipidic cubic phases, for example by using NLP–protein complexes, rather than detergent-solubilized proteins, to feed the cubic phase matrix. Furthermore, the asymmetry of the membrane bilayer is a variable that has not been examined extensively. For example, in the plasma membranes of eukaryotic cells, phosphatidylcholine and sphingomyelin predominate in the outer leaflet, whereas aminophospholipids are primarily located in the cytosolic face. The so-called asymmetric droplet interface technique, combined with in vitro protein synthesis, might be a feasible way to provide a working solution for those proteins that require membrane asymmetry for proper function, such as many ion channels [55]. In the area of synthetic biology, cell-free expression of membrane proteins has an important role. Current challenges in this field include the construction of an enclosed space to accommodate the minimum ingredients for life and the sustainment of membrane-protein production. Researchers have begun to address these problems via the reconstitution of vesicle bioreactors that encapsulate the translation machinery (for examples, see Refs [56,57]). Now, several laboratories are aiming at a full reconstruction of the membrane biogenesis pathway based on in vitro synthesis of functional enzymes inside liposomes. NLPs resemble, in a way, lipidic bicelles, which are mixtures of aliphatic long- and short-chain lipids with a fairly versatile morphology that depends on their composition, temperature and hydration (for a review on lipid bicells, see Ref. [58]). Due to their characteristics, such as their self-alignment in the magnetic field, they have become a valuable tool for applications in the NMR field, and it would certainly be interesting to address the potential advantages of their use in the context of a cell-free protein expression. A general drawback of all cell-free protein expression strategies is their elevated reagent costs. Significant efforts have been made to reduce the amounts of required expensive substrates. For example, the Swartz group has developed a method that uses glucose and nucleoside
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monophosphates as the sources of energy and nucleotides, respectively, thereby lowering the cost by over 75% [59]. However, further advances are needed to equalize the cost of cell-free approaches with those of the more economical cell-based methods. Finally, major efforts are being made to produce in vitro therapeutic drugs, such as G-protein-coupled receptors (GPCRs) (for further information on GPCRs, see Ref. [60]). Here, the limitations of current cell-free approaches are their poor efficiency in achieving proper folding and activity of these high-value proteins. This difficulty is particularly daunting when considered together with the fact that the overproduced protein requires accurate posttranslational modifications, such as glycosylation. Solving these and other currently unanticipated challenges will occupy researchers for several years to come, but at the pace this technology is expanding, we anticipate further exciting breakthroughs in the near future. Acknowledgements We gratefully acknowledge Sanjay Vasu for commenting upon the manuscript. Life Technologies commercializes cell-free protein expression products under the trademarks of ExpresswayTM and MembraneMaxTM.
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Membrane protein expression: no cells required Federico Katzen, Todd C. Peterson and Wieslaw Kudlicki Life Technologies, 5791 Van Allen Way, Carlsbad, CA 92008, USA
Structural and functional studies of membrane proteins have been severely hampered by difficulties in producing sufficient quantities of properly folded protein products. It is well established that cell-based expression of membrane proteins is generally problematic and frequently results in low yield, cell toxicity, protein aggregation and misfolding. Owing to its inherent open nature, cell-free protein expression has become a highly promising tool for the fast and efficient production of these difficult-toexpress proteins. Here we review the most recent advances in this field, underscoring the potentials and weaknesses of the newly developed approaches and place specific emphasis on the use of nanolipoprotein particles (NLPs or nanodiscs). Introduction Despite the extraordinary differences among organisms belonging to each of the three domains of life, nearly a third of their gene products, in all cases, are destined to be inserted into a lipid bilayer. These products often form supramolecular complexes that operate in diverse and often essential ways. Examples of their biological activities include, but are not limited to, energy generation and conversion in the respiratory chain, cell division, signal transduction and transport processes. Perturbation of their biological activities is one of the primary responses of the cell against infections or in genetic disorders such as cancer. These and other reasons have put membrane proteins in the spotlight of the drug development industry, a statement that is reflected by the fact that they account for over 50% of the current pharmaceutical targets [1]. The natural abundance of membrane proteins is usually too low to purify sufficient material for functional and structural studies. Furthermore, in vivo overexpression of these types of proteins is notoriously problematic, often resulting in low yield, cell toxicity, protein aggregation and misfolding. These hindrances are frequently the consequence of processes that affect cell viability, such as membrane overcrowding or the shortage of the insertion machinery (i.e. the SEC translocon, see Glossary) required for the expression of essential proteins. Another difficulty is that membrane proteins are naturally embedded in a complex and dynamic lipid bilayer, which limits the use of many standard biophysical techniques and further complicates their purification and handling. For example, reconstituting purified membrane proteins into a bilayer support is not a trivial endeavor [2,3]. For a fairly complete picture of the challenges that Corresponding author: Kudlicki, W. ([email protected])
the study of membrane proteins poses, we recommend consulting a seminal review in this area [4]. Cell-free protein expression systems are increasingly being considered as viable alternatives for overcoming the above obstacles, as cell viability and protein expression processes are virtually decoupled in these systems (for indepth reviews on cell-free expression technologies, readers should consult Refs [5–12]). In addition, the open nature of the system allows direct access to the reaction conditions allowing, for example, the supply of accessory reagents, such as lipids (in various forms), folding catalysts or a variety of amphiphilic molecules (Figure 1 and see below). The expansion this field has experienced over the past decade has resulted in significant improvements in the yield, solubility and quality of the membrane proteins. For example, progress made in this area has recently led to the publication of the first crystal structure of a membrane protein produced in vitro [13]. Here, we almost exclusively discuss the most recent advances in this field. For earlier accomplishments, we encourage the reader to consult previous reviews [14–17]. This manuscript primarily focuses on the crucial aspects of the reaction that need to be considered to maximize yield and to facilitate downstream applications of membrane proteins expressed in vitro. Cell-free protein expression in the presence of detergents Perhaps the most obvious strategy for facilitating the expression of membrane proteins in vitro is through the Glossary Apolipoprotein: any of various proteins that combine with a lipid to form a lipoprotein and are a constituent of cholesterol-carrying molecules. Asymmetric droplet interface bilayer: membrane formed by joining lipidmonolayer-encased aqueous droplets that are submerged in an oil–lipid mixture. Critical micellar concentration: concentration of surfactants above which micelles are spontaneously formed. Inclusion body: nuclear or cytoplasmic aggregate of stainable substances, usually proteins. Liposome: a closed lipid vesicle surrounding an aqueous interior. Nanolipoprotein particle (or nanodisc): a discoidal particle of defined and controllable size formed by a phospholipid bilayer planar segment surrounded by a protein coat (scaffold protein). Phase transition: the transformation of a thermodynamic system from one phase to another. At the phase-transition point, physical properties may undergo abrupt change. Proteoliposome: a liposome into which one or more proteins have been inserted, usually by artificial means. Synthetic biology: an area of biological research that combines science and engineering in order to design and build (‘synthesize’) novel biological functions and systems. Translocon: protein complex associated with the translocation of nascent polypeptides across membranes.
0167-7799/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2009.05.005
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Figure 1. Current strategies for the cell-free expression of membrane proteins. The figure shows the key reagents and products involved in each of the approaches further described in the text. For simplicity, reactants intrinsic to the extract as well as buffer are omitted. The gene encoding for the membrane protein is present in all strategies, whereas the associated reagents, such as detergents, liposomes, NLPs or the scaffold-encoding gene, vary depending on the particular approach. (a) Reaction in the presence of detergents. If no additional reagents are added, the protein will be associated with detergent micelles. Alternatively, proteoliposomes can be reconstituted from the detergent-protein micelles, as indicated in the text. (b) Expression of membrane proteins as a precipitate. Proteoliposomes can be reconstituted by solubilizing the protein precipitate with detergent and liposomes. Alternatively, the protein might be solubilized in detergent micelles upon the addition of a mild detergent. (c) Expression in the presence of liposomes or vesicles. Here, liposomes are added directly to the reaction. (d) Expression in the presence of pre-fabricated NLPs. (e) Co-expression with scaffold proteins. Both target and scaffold proteins are expressed in vitro and protein–NLP complexes are then formed in situ. Components shown are not drawn to scale.
addition of detergents to the reaction vessel (Figure 1a). Several publications have addressed the effect of detergents on the productivity, solubility and activity of membrane proteins expressed in vitro. Although the final outcome strongly depends on the protein being expressed, a few commonalities are apparent from the published literature (for comprehensive lists, see Refs [14,18]). Successful experiments in terms of yield and solubilization were those that used mild detergents with relatively low critical micellar concentration (CMC), such as polyoxyethylene-alkyl-ethers (e.g. Brij 35, Brij 58 and Brij 78), digitonin, 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) and n-dodecyl-b-D-maltoside (DDM) [19–22]. As a general rule, to prevent the formation of aggregates or inhomogeneous proteomicelles, the molar ratio between the expressed protein and the micelles must never be higher than 1.0. Detergents with relatively high CMC are generally not recommended because they must be present in rather high concentrations, therefore inhibiting transcription and translation [18]. However, a few exceptions to this rule have been reported, as in the case of torque-generating proteins (such as Vibrio alginolyticus PomA and PomB), which were expressed in the presence of the detergent CHAPS [23]. Furthermore, small proteins such as efflux transporters have been demonstrated to be more tolerant to a wider range of detergents, including polyethylene glycol derivatives such as Triton X-100 [24] or fluorinated surfactants [25]. 456
The major shortcoming of the use of detergents to solubilize membrane proteins is the uncertainty of how well micelle structures mimic the natural membrane protein environment. For example, the absence of a bilayer might have adverse consequences on the structure and activity of proteins [4]. In addition, detergents are denaturants per se, making it difficult to study protein macromolecular structure and function. Overall, detergents provide a convenient means of solubilizing and handling membrane proteins during in vitro synthesis. However, a method of maintaining membrane proteins in a native-like bilayer would greatly extend the accuracy and facility of membrane protein enzymology. Reconstitution or direct insertion into vesicles and liposomes An ideal situation for in vitro membrane protein reconstitution would be to work in an environment that more closely resembles the natural lipid bilayer. A few strategies are being developed to achieve this aim. One of them relies on the incorporation of the membrane protein into liposomes (Figure 1a,b). This approach requires, as a starting point, that the membrane protein has been purified and solubilized (not necessarily in its active form) in detergent micelles. The virtual absence of hydrophobic compartments in a cell-free environment promotes precipitation of hydrophobic proteins, which, in contrast to proteins present in inclusion bodies, can be quickly and effectively
Review solubilized in mild detergents (Figure 1b) [14,26]. In particular, 1-myristoyl-2-hydroxy-sn-glycero-3-[phosphorac-(1-glycerol)] (LMPG) was found to be extremely efficient in solubilizing this type of precipitate [18,27]. Therefore, making membrane proteins insoluble in vitro will facilitate their purification and might constitute a suitable starting point for their reconstitution into liposomes. However, reconstitution experiments must take place under non-equilibrium conditions and so are difficult to stage. Furthermore, no single strategy was shown to serve equally well for all membrane proteins [28] and, consequently, the protocol has to be adjusted for each protein studied. Reconstitution examples of membrane proteins expressed in vitro include those based on the freeze–thaw technique [26,27,29] or using detergent adsorbent particles in the presence of liposomes [30,31]. The freeze–thaw technique is based on the demonstration that freeze–thawing of liposomes mixed with protein enhances the formation of proteoliposomes. Liposomes can be made from a variety of lipids, such as phosphatidylserine, phosphatidylcholine, cholesterol, E. coli lipids or asolectin. Another strategy is to perform the cell-free reaction in the presence of unilamellar liposomes (Figure 1c). Although the mechanism by which proteins are inserted into naked membranes remains elusive, it is apparent that the insertion efficiency is strongly dependent on the phase transition temperature, the length of the lipid and the overall hydrophobic thickness of the bilayer [32]. Successful expression of a variety of functional proteins, such as stearoyl-CoA desaturase, bacteriorhodopsin, apoCytochrome b5 and the catalytic core of the NADPH oxidase complex, has been reported using liposomes made of a variety of lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), phosphatidylcholine, soybean lipids and spinach thylacoids [32–36]. Some of these functional proteoliposomes have been identified as good candidates for the delivery of therapeutic proteins [35,36]. A disadvantage of working with unilamellar liposomes is the relative heterogeneity of the samples and the presence of residual multilamellar vesicles [37]. This feature prevents the application of this strategy in structural biology studies. For example, liposome particles ranging widely in size between 30 and 200 nm are commonly obtained using currently established protocols [15]. Although procedures for preparing more homogeneous liposome populations exist [38], these require unusual equipment and are not widely used. Another approach stems from the rationale that most of the membrane proteins in Gram-negative bacteria are targeted to their final location by processes that depend on the signal recognition particle (SRP) receptor, the SecYEG translocon and YidC, which are components embedded in the natural target membranes [39]. These components can be added to the reaction as part of inverted vesicles made from E. coli cells (Figure 1c), as proposed by Kuruma and coworkers [40]. These authors reconstituted the E. coli membrane integration and translocation mechanisms using a defined cell-free system that was supplemented with E. coli inverted vesicles and revealed that no cytosolic factors other than the SRP and SecA are required
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for membrane protein integration [40]. More recently, Wuu and Swartz reported the high-yield expression of functional integral membrane proteins using an energy-efficient cell-free system supplemented with E. coli inner membrane vesicles [41]. Although this approach addresses the expression of Sec- and SRP-dependent proteins, it relies on the availability of high-quality inverted E. coli vesicle samples, which are difficult to prepare and, to our knowledge, not commercially available. By contrast, crude vesicles derived from dog pancreas have been commercialized for over two decades; however, they have a profound negative effect on the translational rate of the resulting lysate [42]. Finally, the relatively high heterogeneity of vesicle preparations in general might preclude using reaction products embedded in these structures for structural studies. This drawback is resolved by new technologies, as discussed below. Use of nanolipoprotein particles To overcome the shortcomings stemming from the use of microsomes and vesicles, novel strategies based on the use of ‘nanodiscs’ or ‘nanolipoprotein particles’ (NLPs) have been recently proposed (Figure 1d,e). NLPs are nanometersized, discoidal particles comprising amphipathic helical proteins (scaffold proteins) that wrap themselves around the planar circumference of a lipid bilayer (for a fairly complete review on NLPs, see Ref. [37]) (Figure 2a). A variety of scaffold proteins, such as apolipoproteins A1, E and C and insect lipophorins, have been shown to spontaneously self-assemble into NLPs in the presence of phospholipids [43,44]. Contrary to what is observed with liposomes or vesicles, NLP preparations are remarkably monodisperse (molecular mass deviation <5%) owing to the scaffold protein being the major determinant of their average diameter (9 to 20 nm depending on the type of scaffold) [43–45]. Other advantages of NLPs compared with liposomes include: (i) better representation of the phase transition behavior of biological membranes [37]; (ii) simplicity of preparation (no unconventional instrumentation required); and (iii) unrestricted access to both faces of the lipid bilayer. One of the cell-free NLP approaches for producing membrane proteins makes use of preformed (empty) NLPs that are added to the cell-free reaction and thus serve as a membrane support for the membrane protein [46,47] (Figure 1d). The products of the reaction are NLP–protein complexes, empty NLPs and residual precipitated membrane proteins. Virtually pure soluble complexes can be readily purified by means of a tag fused to the membrane protein (precipitated material is removed by a short precentrifugation step) or, even more stringently, by tandem purification using a second peptide tag fused to the scaffold protein. A comprehensive expression study of many biologically important membrane proteins of different topologies, origin, sizes and proposed roles showed that, without exception, all tested proteins dramatically increased their solubility in the presence of NLPs [46,47] (Figure 2c). Importantly, analytical assays for a subset of these model membrane proteins indicated that the proteins were also correctly folded and active (for an example, see Figure 2b). The approach has been validated using cell-free extracts 457
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Figure 2. Cell-free protein expression in the presence of NLPs. (a) Illustration of a complex between an NLP and a membrane protein. Transmembrane segments (a helices) are shown in red, and the outer gray shell represents the scaffold protein ring. (b) Activity of the E. coli multidrug transporter EmrE expressed in vitro. Radioactive tetraphenylphosphonium (TPP+)-binding was determined. Binding constants are in agreement with previous determinations [46]. As shown in the inset, [3H]TPP+ binding was performed either in the absence (empty bars) or presence (filled bars) of nonradioactive TPP+. Binding reactions were carried out with EmrE synthesized in the presence (+) or absence ( ) of NLPs, demonstrating that enzymatic activity was only obtained in the presence of NLPs. Adapted from [46]. (c) Solubility of a set of proteins expressed either in the presence (filled bars) or absence (empty bars) of NLPs. In the presence of NLPs, protein solubility was significantly increased. For the analyzed dataset, the overall solubility increased from 17.3 2.2% to 78.8 3.4%. GenBank accession numbers and number of predicted transmembrane segments (TMSs) of their protein products are indicated. Black, red and green annotations identify the gene source as human, murine and bacterial, respectively [46]. Reproduced from [46] with the permission of ACS Publications.
derived from wheat germ, rabbit reticulocytes and E. coli cells [46]; however, only the latter has been consolidated into a commercially available kit [48] (MembraneMaxTM Protein Expression Kit, Life Technologies). Another advantage of the system is that, as opposed to the above-mentioned pancreatic dog microsomes, NLPs exhibit no detrimental effects on the translation efficiency of the lysates [46]. Yield is protein dependent and linearly scalable, topping at 0.9 mg/ml of soluble product in the case of bacteriorhodopsin [46]. The main limitation to this approach, as with the liposomes, relates to the absence of a translocon (and also a transmembrane potential), which is required for the proper insertion and folding of a variety of integral membrane proteins [39,49]. However, reconstitution of the bacterial SecYEG complex into NLPs has been successfully attained [50,51], setting the scene for an assisted-insertion mechanism of Sec-dependent proteins into the particles. Finally, it is worth mentioning that the NLP–protein association process in the absence of a translocon is believed to be cotranslational, as no complexes are formed after blocking the peptide chain 458
elongation activity of the extract [48]. To insert a pre-made protein into the NLPs, a reconstitution approach should be followed [44]. A second cell-free NLP strategy stems from the ability of apolipoproteins (fat-bound proteins) to sequester lipid bilayer patches in solution. In this approach, the scaffold protein is co-expressed with the target membrane protein in the presence of liposomes, resulting in the in situ assembly of empty and target-filled NLP structures [52] (Figure 1e). The benefit of this method is its inherent simplicity, as there is no need to produce NLPs before the assay. However, phospholipids need to be processed in advance to obtain suitable unilamellar liposomes. The drawback of this method is that the NLP–protein complex has to be purified out of a large set of reaction products consisting of liposomes, proteoliposomes and lipoprotein particles of varying sizes. In addition, the ratio between the two programming DNA templates (coding for the scaffold and target proteins) has to be adjusted for each individual protein target. This approach has been validated for batch and continuous exchange formatted reactions [52]. Yields
Review are considerably lower than when using pre-assembled NLPs, as an important fraction of the resources has to be dedicated to the expression of the scaffold protein. The methods described above highlight the need to distinguish particles that can differ by as little as a fraction of a nanometer (in height). For that purpose, atomic force microscopy has proven to be a very robust technique, allowing the rapid scanning of a surface containing hundreds of particles and reliably discriminating between empty and filled NLPs [45,53]. Future perspectives Although cell-free protein expression has already proven to be an extraordinary vehicle for studying membrane proteins, it still faces major challenges. For example, an area within this technology that still warrants further improvement is structural biology. Although some structures have been resolved using X-ray crystallography and solid state NMR [13,54], a successful outcome is not the typical case (for a review see Ref. [54]). It would be interesting, for instance, to examine the synergies between the cell-free-NLP approach and crystallization in lipidic cubic phases, for example by using NLP–protein complexes, rather than detergent-solubilized proteins, to feed the cubic phase matrix. Furthermore, the asymmetry of the membrane bilayer is a variable that has not been examined extensively. For example, in the plasma membranes of eukaryotic cells, phosphatidylcholine and sphingomyelin predominate in the outer leaflet, whereas aminophospholipids are primarily located in the cytosolic face. The so-called asymmetric droplet interface technique, combined with in vitro protein synthesis, might be a feasible way to provide a working solution for those proteins that require membrane asymmetry for proper function, such as many ion channels [55]. In the area of synthetic biology, cell-free expression of membrane proteins has an important role. Current challenges in this field include the construction of an enclosed space to accommodate the minimum ingredients for life and the sustainment of membrane-protein production. Researchers have begun to address these problems via the reconstitution of vesicle bioreactors that encapsulate the translation machinery (for examples, see Refs [56,57]). Now, several laboratories are aiming at a full reconstruction of the membrane biogenesis pathway based on in vitro synthesis of functional enzymes inside liposomes. NLPs resemble, in a way, lipidic bicelles, which are mixtures of aliphatic long- and short-chain lipids with a fairly versatile morphology that depends on their composition, temperature and hydration (for a review on lipid bicells, see Ref. [58]). Due to their characteristics, such as their self-alignment in the magnetic field, they have become a valuable tool for applications in the NMR field, and it would certainly be interesting to address the potential advantages of their use in the context of a cell-free protein expression. A general drawback of all cell-free protein expression strategies is their elevated reagent costs. Significant efforts have been made to reduce the amounts of required expensive substrates. For example, the Swartz group has developed a method that uses glucose and nucleoside
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monophosphates as the sources of energy and nucleotides, respectively, thereby lowering the cost by over 75% [59]. However, further advances are needed to equalize the cost of cell-free approaches with those of the more economical cell-based methods. Finally, major efforts are being made to produce in vitro therapeutic drugs, such as G-protein-coupled receptors (GPCRs) (for further information on GPCRs, see Ref. [60]). Here, the limitations of current cell-free approaches are their poor efficiency in achieving proper folding and activity of these high-value proteins. This difficulty is particularly daunting when considered together with the fact that the overproduced protein requires accurate posttranslational modifications, such as glycosylation. Solving these and other currently unanticipated challenges will occupy researchers for several years to come, but at the pace this technology is expanding, we anticipate further exciting breakthroughs in the near future. Acknowledgements We gratefully acknowledge Sanjay Vasu for commenting upon the manuscript. Life Technologies commercializes cell-free protein expression products under the trademarks of ExpresswayTM and MembraneMaxTM.
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