Tables for
Volume F
Crystallography of biological macromolecules
Edited by E. Arnold, D. M. Himmel and M. G. Rossmann

International Tables for Crystallography (2012). Vol. F, ch. 3.2, pp. 92-98
doi: 10.1107/97809553602060000811

Chapter 3.2. Expression and purification of membrane proteins for structural studies

J. A. Ernst,a,b D. G. Yansurac and C. M. Kothd*

aDepartment of Protein Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, USA,bDepartment of Protein Engineering, Genentech, 1 DNA Way, South San Francisco, California 94080, USA,cDepartment of Antibody Engineering, Genentech, 1 DNA Way, South San Francisco, California 94080, USA, and dDepartment of Structural Biology, Genentech, 1 DNA Way, South San Francisco, California 94080, USA
Correspondence e-mail:

Despite the fact that over 200 unique membrane-protein structures have now been solved, significant challenges remain at all stages of protein production for both structural and functional studies. First, recombinant expression levels of membrane proteins are typically very low. Second, target proteins must be solubilized from their native environment, the cell membrane, and purified. This requires the use of detergents that can also cause the protein to denature or aggregate. Indeed, the repertoire of detergents that have been used successfully for membrane-protein structural studies is surprisingly limited. Third, obtaining well ordered crystals is often very difficult, even if milligramme quantities of high-quality membrane protein can be obtained. Despite these and other barriers, significant progress has been made over the last several years in determining the structures of members of many challenging membrane-protein families, including transporters, channels, intramembrane proteases and G-protein-coupled receptors. Based largely on these past successes, and analyses of recurrent trends, reasonable `first-pass' strategies can now be proposed for most membrane-protein targets. Here, the current state of producing membrane proteins for structural studies is reviewed. An evidence-guided approach is detailed that should provide reasonable starting points for the production of many membrane-protein targets.

3.2.1. Introduction

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Integral membrane proteins constitute about a third of the proteome of most organisms but less than 1% of all entries in protein structural databases (Berman et al., 2002[link]). This disparity is largely due to inherent difficulties in their expression, solubilization and purification. The production of sufficient protein for structural studies can be challenging for any target, but several obstacles are unique to membrane proteins. For example, proper insertion into the membrane relies on host cellular machinery that may be limiting or incompatible. Exceeding this capacity can lead to cell death or the accumulation of aggregated and inactive protein within the cell (Geertsma et al., 2008[link]). Also, post-translational modifications such as glycosylation, acylation and sulfation may not be faithfully reproduced (Grisshammer & Tate, 1995[link]). As a further complication, even if sufficient expression can be achieved, most membrane-protein structural studies require that the target be extracted from the cellular membrane using detergents. However, detergents can adversely affect protein structure and function, as well as influence the outcome of crystal trials (Engel et al., 2002[link]; Lemieux et al., 2003[link]; Prive, 2007[link]). Fortunately, if a strategy for purifying sufficient quantities of a given membrane-protein target can be established, crystallization strategies largely mimic the standard techniques for soluble proteins (Newby et al., 2009[link]).

Despite the aforementioned challenges, high-resolution structures of almost 200 unique membrane proteins have now been solved ( ), the vast majority using protein produced by recombinant methods (Willis & Koth, 2008[link]). The rate of new structure determinations has also increased dramatically over the last few years, mimicking the exponential growth of soluble protein structures in the early 1980s. Space limitations here preclude a thorough review of all possible membrane-protein production methods. However, an examination of the successful expression, solubilization and purification strategies that have led to membrane-protein structures reveals that, in many cases, remarkably similar methods have been used (Carpenter et al., 2008[link]; Willis & Koth, 2008[link]; Newby et al., 2009[link]). Guided by these methods, the following sections detail a rational and consensus `first-attempt' strategy that has worked for a broad range of membrane targets with only minor variations in technique (Dobrovetsky et al., 2005[link], 2007[link]; Lunin et al., 2006[link]). It must be noted that while this represents an evidence-guided approach, the methods provided herein will not work for every membrane protein and, in fact, will fail for many. Thus, prioritized lists of alternative strategies are provided for those targets in which the initial expression or isolation attempts do not succeed or problems are encountered.

3.2.2. A consensus strategy for membrane-protein expression

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Historically, the likelihood of successful structure determination has been high for those membrane proteins that can be isolated from readily available abundant natural sources (Sakai & Tsukihara, 1998[link]). Unfortunately, most membrane proteins do not meet this criterion. In reality, the vast majority of high-resolution membrane-protein structures are of prokaryotic targets, expressed in Escherichia coli by recombinant methods (Willis & Koth, 2008[link]; Newby et al., 2009[link]). The reasons for this are simple. Attempting recombinant expression in E. coli is inexpensive, flexible, simple and easily scaled-up, and many constructs and strains can be screened quickly. As with crystallographic efforts for many soluble proteins (Gräslund et al., 2008[link]), most successful membrane-protein endeavours also commonly screen multiple constructs and/or orthologues for any given target (see, for example, Chang et al., 1998[link]; Doyle et al., 1998[link]), since the greater the number of unique constructs screened, the greater the chance that one will be successfully isolated and crystallized. Given the overwhelming use of E. coli for successful membrane-protein structures, a `first-pass' expression strategy suitable for almost any membrane protein is clear: attempt expression in E. coli and, whenever possible, screen multiple constructs, orthologues and strains. Choosing the expression system and affinity tags

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In addition to the choice of expression host, one must also consider the expression system and the type and placement of affinity tags or fusion proteins. For E. coli, IPTG-inducible T7 polymerase-driven expression systems, such as those based on pET vectors (Studier & Moffatt, 1986[link]) and λDE3 lysogen strains, are the most widely used for membrane proteins, as is the case for soluble targets. Also, it has generally been observed that for most targets, protein expression is optimal at lower temperatures (i.e. <20 °C; Christendat et al., 2000[link]; Wang et al., 2003[link]; Dobrovetsky et al., 2005[link]). When structural studies are the desired outcome, the most common tagging strategy for membrane proteins is to engineer a stretch of at least six histidine residues at the amino or carboxyl terminus of the target constructs; this is used for >80% of successful targets (Willis & Koth, 2008[link]). If expression levels are sufficient (i.e. ≥0.05 mg g−1 cell paste), this often permits purification using a general two-step procedure: capture of the tagged protein by immobilized metal affinity chromatography (IMAC), followed by size-exclusion chromatography (SEC) (see below). Remarkably, this basic approach has proved successful for the crystallization of many membrane proteins, as discussed in the following section. If premature termination is observed during expression, the engineering of a carboxyl-terminal tag will ensure that these proteins are not isolated during purification. Also, extending the stretch of histidines to greater than six residues can improve the retention of membrane proteins on immobilized metal affinity resins. This can prove particularly useful, given the modest expression levels and reduced chromatographic resolution and recovery of many membrane targets (Dobrovetsky et al., 2005[link]; Eshaghi et al., 2005[link]; Surade et al., 2006[link]; Lewinson et al., 2008[link]). Other affinity tags are viable options, but are more rarely used. For example, although fusion proteins such as glutathione S-transferase (GST) and thioredoxin are widely used to promote expression and/or simplify the purification of soluble proteins [for an extensive comparison of various affinity tags, see Lichty et al. (2005[link])], they are rarely cited in expression strategies for polytopic membrane proteins. Strategies for improving membrane-protein expression

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Low expression levels are common for many membrane proteins. Before testing alternative hosts, it may be worthwhile first to screen alternative E. coli expression systems, such as those using the tightly controlled arabinose promoter (Guzman et al., 1995[link]). Also, other E. coli strains can be tested. For example, the mutant C41 and C43 `Walker' expression strains (Miroux & Walker, 1996[link]; Wagner et al., 2008[link]) yield higher expression levels for some membrane proteins. Table[link] summarizes common strategies for improving the expression of membrane proteins in E. coli. If reasonable efforts in E. coli still do not yield sufficient expression, then an alternative expression host should be considered. The next most successful is yeast. Of the 22 recombinant eukaryotic membrane proteins whose structures have been solved by crystallography, half were produced using Pichia pastoris (seven targets), Saccharomyces cerevisiae (three targets) or Saccharomyces pombe (one target) (Raman et al., 2006[link]). Membrane proteins expressed in yeast are typically fused to an amino-terminal host signal sequence to promote proper mem­brane targeting. The use of higher eukaryotic hosts, such as insect and mammalian cells, is also increasing as more groups attempt to crystallize recombinant eukaryotic targets. The recent high-resolution structures of several G-protein-coupled receptors are notable examples of targets expressed successfully in insect cells (Cherezov et al., 2007[link]; Rasmussen et al., 2007[link]; Hanson et al., 2008[link]; Jaakola et al., 2008[link]; Warne et al., 2008[link]).

Table| top | pdf |
Strategies for improving recombinant membrane-protein expression in E. coli

Troubleshooting expression problems: if the `first-pass' expression strategy is unsuccessful, then some changes in the expression construct, host strain or induction conditions may be helpful. The symptoms of the problem often give a clue as to how to make these corrections.

Symptom Solution
E. coli colonies small after transformation into expression strain Need tighter promoter such as phoA, araC etc.
Unprocessed signal sequence If none is used in native situation, remove from construct
  If one is present in native situation, use an expression host co-translational signal sequence
  If still unprocessed, lower translation initiation strength
Mostly high-molecular-weight aggregates Decrease induction time
Rapid cell growth after induction with low expression levels Increase translation initiation strength
Poor cell growth after induction with low expression levels  
 (a) Accumulation maximum after only 1–3 h Decrease translation initiation rates or plasmid copy number
 (b) Accumulation increases out to 8–10 h post induction Usually successful:
   (a) Re-synthesize the gene, removing rare codons and optimizing codon pairs
   (b) Convert transmembrane and intracellular domain cysteines (normally reduced) to serines
   (c) Try different non-DE3 lysogen strains after switching to a non-T7 promoter
    (i) Cytoplasmic protease deletions
    (ii) Periplasmic protease deletions
    (iii) Heat-shock htpRTS
  Sometimes successful:
   (a) Try different C-terminal tags such as poly-his, Flag etc.
   (b) Try different temperatures of induction such as 20, 25, 30 °C
These methods are also useful for optimizing eukaryotic gene expression.

To date, there is only one report of a structure determined from cell-free synthesis of the target membrane protein (EmrE; Chen et al., 2007[link]). However, for several reasons, cell-free approaches should be explored as a complement to traditional expression methods. Firstly, the absence of a host cell negates toxicity problems for some target proteins. Secondly, many targets that do not express in cells can be produced cell-free (Ishihara et al., 2005[link]; Klammt et al., 2007[link]; Savage et al., 2007[link]). Thirdly, these systems allow for considerable flexibility with respect to reaction conditions and components. Cell-free systems for prokaryotic and eukaryotic hosts have been described (Schwarz et al., 2008[link]), some of which function in the presence of solubilizing detergents (Ishihara et al., 2005[link]; Klammt et al., 2007[link]; Savage et al., 2007[link]) or lipids (Kuruma et al., 2005[link]; Schwarz et al., 2007[link]), facilitating proper folding and downstream purification of membrane-protein targets.

Many eukaryotic proteins have undergone extensive construct engineering to improve expression and/or crystallization outcomes. These include fusion to T4 lysozyme, mutation of glycosylation sites, deletion of potentially disordered regions and/or targeted evolution to identify conformationally stable mutants (Long et al., 2005[link]; Cherezov et al., 2007[link]; Rosenbaum et al., 2007[link]; Jaakola et al., 2008[link]; Magnani et al., 2008[link]; Serrano-Vega et al., 2008[link]; Warne et al., 2009[link]). Also worth mentioning is the observation that incorporation of an amino-terminal `rhodopsin tag' comprising the first 20 amino acids of bovine rhodopsin (amino-acid sequence MNGTEGPNFYVPFSNKTGVV) has been found to boost dramatically the expression levels of eukaryotic membrane targets expressed in insect and mammalian cells (Krautwurst et al., 1998[link]).

3.2.3. A consensus strategy for membrane-protein purification

| top | pdf | General principles

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The purification of an expressed integral membrane protein for structural studies typically involves four discrete steps: cell lysis, membrane isolation, detergent extraction and chromatographic separation. In this section, an evidence-based consensus strategy is outlined which aims to serve as a starting point for membrane-protein purification. Detailed protocols are outlined in Fig.[link]. Note that the most significant complication in this process is the need to use detergents in all steps of protein handling and chromatography. Detergents permit the extraction of target proteins from the mem­brane, but they must also maintain protein stability and activity by mimicking, as closely as possible, the lipid bilayer. Although many detergents do meet these criteria, relatively few are compatible with high-resolution structural studies.


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Flow diagram of the membrane-protein purification strategy. Typically, only two chromatographic steps (affinity chromatography and SEC) are required for proteins that are expressed at reasonably high levels with a hexa-histidine tag. Ion-exchange chromatography (IEC) methods are sometimes employed, although they are not as common as SEC. If no crystals are obtained in initial sparse matrix screens, the protein can be exchanged to a different detergent and crystal trials attempted again (dotted arrow). Suggested `first-try' buffers are indicated at each step. Cell lysis and membrane isolation

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Cellular lysis methods for the isolation of integral membrane proteins are similar to those for soluble targets. Cells over-expressing the protein of interest are re-suspended in a lysis buffer lacking detergent. Typically, high-flow/high-pressure cell-disruption devices are used, such as a continuous-flow homogenizer (EmulsiFlex, Avestin, Canada) or microfluidizer (Microfluidics Inc., Newton, Massachusetts, USA). Although these two devices achieve lysis by different mechanisms, both can disrupt the cells of almost all commonly used expression hosts, including yeast, and permit the rapid processing of large volumes. Other cell-disruption methods, such as sonication or nitrogen cavitation, can also be used for eukaryotic cells and, with slightly lower efficiency, E. coli. Protease inhibitors are included at this stage, although they are often not necessary in the later stages of purification. Following lysis, the membrane fraction of the cells is pelleted by ultracentrifugation. Detergent extraction of membrane proteins

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Following centrifugation, the crude-membrane pellet is re-suspended in a native lysis buffer. Detergent is then added to extract the membrane proteins from the lipid bilayer, at a molarity well above the critical micelle concentration (CMC, the concentration above which detergent molecules aggregate to form micelles). It is also more common than not that the same detergent used for solubilization is also used for subsequent purification and crystallization (Willis & Koth, 2008[link]), albeit at a much lower concentration (typically just above the CMC). Broadly speaking, detergents fall into three main categories: charged (ionic), zwitterionic and non-charged (polar). Detergents containing no net charge are more likely to solubilize membrane proteins in their native state and are therefore more widely used for structural studies. In addition, detergents with sugar residues as head groups have proved particularly successful for the crystallization of membrane proteins, as have polyoxyethylene monoalkylethers (CnEm; Prive, 2007[link]; Newstead et al., 2008[link]). Of all the membrane-protein structures solved to date, almost 70% have used one of just six different detergents for solubilization (Newstead et al., 2008[link]; Willis & Koth, 2008[link]); with the exception of dodecyl-N,N-dimethylamine N-oxide (LDAO), all are non-charged. These six are, starting with the most prevalent, dodecyl-β-D-maltopyranoside (DDM), LDAO, octyl-polyoxyethylene (C8POE), decyl-β-D-maltoside (DM), octyl-β-D-glucoside (OG) and Triton X-100. Of these, DDM has been used for one out of almost every four crystallized targets and should be tested with any new membrane protein. The general properties of detergents and their use for membrane-protein solubilization have been reviewed by Hjelmeland (1990[link]) and Neugebauer (1990[link]).

The ability of detergents to solubilize target proteins can be tested on a small scale. The concentration of detergent that is optimal for solubilization should be determined empirically, although this can quickly become a multidimensional problem. A simple starting strategy, based on the purification methods for successfully crystallized membrane proteins, is to screen the aforementioned detergents at 1% (DDM, LDAO, Triton X-100) or 2% (C8POE, DM, OG). Detergents that are effective at these concentrations can be further screened to determine the optimal solubilization conditions (i.e. by varying detergent concentration versus solubilization time). Here, `optimal' refers to the conditions yielding the highest level of soluble active target protein, or, if no activity assay exists, simply the conditions yielding the most target protein (but see caveats below). Although the level of solubilized target protein usually increases with time, many membrane proteins show reduced stability in detergent. It is advantageous, and more common, to minimize solubilization times (i.e. <4 h), thereby facilitating a more rapid purification. Physical methods such as sonication and passage through a cell disruptor may also be used to speed the solubilization process. Target proteins are considered soluble if, in the presence of detergent, they remain in solution after being subjected to 100 000 g for 1 h. General considerations for monitoring membrane-protein activity

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Although often overlooked (surprisingly so), developing a means of following membrane-protein activity can prove critical when optimizing solubilization and purification conditions. Unfortunately, there is no universal method for assaying the activity of all solubilized or purified membrane proteins. For receptors, obvious assays include those based on monitoring ligand binding. For many other targets, such as those critically dependent on the vectorial nature of the membrane for activity (i.e. ion channels), there may be no direct assay in the detergent-solubilized state. On the premise that any assay is better than none, some simple biophysical analyses are worth mentioning. Firstly, some membrane targets that function as multimers have been shown to retain quaternary structure in sodium dodecyl sulfate (SDS) and during polyacrylamide gel electrophoresis (PAGE) (Cortes & Perozo, 1997[link]; Prive, 2007[link]). This observation can provide a convenient means of monitoring quaternary structure under various solubilization or purification conditions. It has also been suggested that proteins demonstrating this behaviour may, in general, be stable in a variety of detergents, as is the case for the KcsA potassium channel (Cortes & Perozo, 1997[link]). Secondly, for those multimeric targets whose quaternary structure is not maintained in SDS, monitoring the degree of cross-linking in various detergents/conditions can provide for a simple, albeit indirect, structural probe (Sukharev et al., 1999[link]). Thirdly, monitoring the sensitivity of a target protein to limiting amounts of protease can also provide a simple means of probing `activity' or structure under various solubilization/purification conditions. For example, binding to magnesium alters the protease sensitivity of detergent-solubilized CorA channels, and this can be easily monitored by SDS–PAGE analysis of digested samples (Payandeh & Pai, 2006[link]). In theory, such an assay could be used to probe structural changes upon ligand binding for almost any target, so long as a ligand is known. The growing use of Fab antibody fragments as aids to membrane-protein crystallization provides a fourth assay for some targets (Hunte & Michel, 2002[link]; Day et al., 2007[link]). Antibodies (or antibody fragments) that bind a target protein in a cellular environment should also be able to bind that protein in detergent, as evaluated by an assay such as immunoprecipitation. Ideally, such antibodies are western-negative (i.e. they recognize nonlinear peptide epitopes that would only be present in a properly folded protein). Lastly, membrane proteins containing disulfide bonds (common in extracellular domains) often migrate more rapidly in SDS–PAGE than the same protein under reducing conditions. This can be used as an assay for proper protein folding in the cell, although this method does not provide information about target-protein stability in detergent. Primary purification: affinity chromatography

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Given that most membrane proteins are cloned with a histidine tag, the most common method for their primary purification is immobilized metal affinity chromatography (IMAC), used in >80% of recombinant membrane-protein structures (Willis & Koth, 2008[link]; Newby et al., 2009[link]). IMAC methods for membrane proteins do not differ significantly from those commonly used for soluble proteins, with the notable exception that detergent is required in all buffers. During the wash and elution steps, the detergent concentration is typically reduced to a level above the CMC that is only just sufficient to maintain target-protein solubility. Ideally, the ratio of micelles to membrane-protein molecules is ∼2 at this and subsequent purification stages [for an in-depth analysis, the reader is strongly encouraged to read Helenius et al. (1979[link]) and Wiener (2004[link])]. The histidine tag may be removed after IMAC elution, although it is also worthwhile to proceed with a sample that retains the tag, since they provide critical lattice contacts in some protein crystals (Carson et al., 2007[link]). Secondary purification: size-exclusion and ion-exchange chromatography

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For most successful structures, target membrane proteins are sufficiently pure for crystal trials after a two-step purification: IMAC followed by size-exclusion chromatography (SEC) or ion-exchange chromatography (IEC) (Willis & Koth, 2008[link]; Newby et al., 2009[link]). A detailed strategy is outlined in Fig.[link]. SEC is the most common secondary (i.e. post-IMAC) method, represented in about half of all successful membrane-protein structures (Willis & Koth, 2008[link]). IEC methods have been used less frequently, being employed for about one out of every four structures (Willis & Koth, 2008[link]). The simple fact that some integral membrane proteins lack sufficient polar area to interact effectively with ion-exchange resins may account for the difference. Both methods permit the exchange of one detergent (i.e. that used for solubilization) for another or to a higher grade (a consideration, given the high cost of many detergents). Ion-exchange methods are likely to give a more complete detergent exchange, although this does not necessarily improve crystallization outcomes (Lemieux et al., 2003[link]). In fact, incomplete detergent exchange and/or incomplete removal of specifically bound lipids can prove beneficial in downstream crystal trials (Long et al., 2007[link]). Concentrating membrane-protein samples for crystallography

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Concentration of the target membrane protein is typically required prior to crystallization. Many different concentration methods can be used (i.e. those employed for soluble proteins), although some caution is warranted. Detergent will normally concentrate with the protein, since most detergent micelles are too large to pass through typical dialysis or concentration mem­branes. This can be detrimental to crystallization; high detergent concentrations can denature protein or limit diffraction (Wiener, 2004[link]). As a general rule, the highest-molecular-weight cutoff membrane that does not permit passage of the target protein should be used, as this will allow the passage of at least some protein-free detergent micelles. Also, any size-exclusion steps should be performed at a relatively high protein concentration (10–20 mg ml−1 or higher) to minimize volumes. Purified protein may also be dialysed to exchange non-detergent buffer components, so long as the detergent concentration of the bulk solution is maintained just above the CMC. However, the dialysis of many detergents will proceed slowly (days to weeks). Accordingly, dialysis does not appear to be a widely used method for controlling detergent concentration prior to crystallization (reviewed by Willis & Koth, 2008[link]).

3.2.4. Common purification pitfalls and prioritized alternative strategies

| top | pdf | Poor solubility of the target protein

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If the target protein does not remain soluble during purification, an obvious alternative strategy is to test different detergents or detergent combinations. Given that just a handful of different detergents have been used to solubilize and crystallize most membrane proteins, testing alternatives is usually not a daunting task. The most common method of detergent exchange is SEC (Fig.[link]).

The zwitterionic lyso-lipid mimetic detergents such as FOS-CHOLINE 12 (dodecylphosphatidylcholine) or 14 (FC12 and FC14; Anatrace, Maumee, Ohio, USA) will solubilize many membrane proteins (Eshaghi et al., 2005[link]) and there are a few reports of their use in crystallography [MscS mechanosensitive channel (Bass et al., 2002[link]); protein-conducting channel (van den Berg et al., 2004[link])]. Should solubilization screening with the more commonly used detergents prove ineffective, it may be possible to solubilize first with a `stronger' more lipid-like detergent, such as FC12, and then exchange to a non-charged detergent, such as DDM, during purification. The isolated target protein does not crystallize

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Although an in-depth analysis of membrane-protein crystallization methods is beyond the scope of this chapter (see instead Chapter 4.2[link] ), there are many purification parameters that can be varied to improve crystallization outcomes should initial trials fail to yield `hits' or well diffracting crystals. Perhaps the foremost method is to exchange the protein into an alternative detergent or detergent mixture, typically by SEC, and repeat the crystal trials. Since the vast majority of membrane-protein structures have used one of only a handful of detergents (see above), it is likely that sampling even just a small number of other detergents would dramatically increase the likelihood of obtaining or optimizing crystals. Also, an increase in the alkyl-chain length by one methylene group (i.e. nonyl- versus octyl-β-D-glucoside) often leads to an increase in protein stability, and this too can improve crystallization outcomes (Wiener, 2004[link]). If significant covalent disulfide aggregation of the protein is observed during purification, alkylation of free cysteines should be considered during the early stages of target-protein isolation. Other parameters that could be varied include the solution pH, salt concentration and temperature, or supplementing with known ligands, to name just a few.

3.2.5. Summary

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There are many possible routes for the expression and isolation of membrane proteins for structural studies, and many factors can affect the likelihood of obtaining diffraction-quality crystals. The daunting prospect for almost any new membrane target is that every stage, including expression, solubilization, purification and crystallization, will prove challenging. In this light, it is encouraging that remarkably similar techniques have been employed for many successfully crystallized membrane targets, including, for example, the observation that most structures have been solved using just a handful of different detergents throughout the isolation and crystallization steps. It is equally significant that simple two-step purification strategies predominate. Clearly, new methods for the production of membrane proteins will continue to be developed. Nevertheless, for a field that is quite likely to be entering an acute phase of growth, a solid foundation has been established for reasonable `first-try' strategies.


Bass, R. B., Strop, P., Barclay, M. & Rees, D. C. (2002). Crystal structure of Escherichia coli MscS, a voltage-modulated and mechanosensitive channel. Science, 298, 1582–1587.
Berg, B. van den, Clemons, W. M., Collinson, I., Modis, Y., Hartmann, E., Harrison, S. C. & Rapoport, T. A. (2004). X-ray structure of a protein-conducting channel. Nature (London), 427, 36–44.
Berman, H. M., Battistuz, T., Bhat, T. N., Bluhm, W. F., Bourne, P. E., Burkhardt, K., Feng, Z., Gilliland, G. L., Iype, L., Jain, S., Fagan, P., Marvin, J., Padilla, D., Ravichandran, V., Schneider, B., Thanki, N., Weissig, H., Westbrook, J. D. & Zardecki, C. (2002). The Protein Data Bank. Acta Cryst. D58, 899–907.
Carpenter, E. P., Beis, K., Cameron, A. D. & Iwata, S. (2008). Over­coming the challenges of membrane protein crystallography. Curr. Opin. Struct. Biol. 18, 581–586.
Carson, M., Johnson, D. H., McDonald, H., Brouillette, C. & DeLucas, L. J. (2007). His-tag impact on structure. Acta Cryst. D63, 295–301.
Chang, G., Spencer, R. H., Lee, A. T., Barclay, M. T. & Rees, D. C. (1998). Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel. Science, 282, 2220–2226.
Chen, Y. J., Pornillos, O., Lieu, S., Ma, C., Chen, A. P. & Chang, G. (2007). X-ray structure of EmrE supports dual topology model. Proc. Natl Acad. Sci. USA, 104, 18999–19004.
Cherezov, V., Rosenbaum, D. M., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., Choi, H. J., Kuhn, P., Weis, W. I., Kobilka, B. K. & Stevens, R. C. (2007). High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science, 318, 1258–1265.
Christendat, D., Yee, A., Dharamsi, A., Kluger, Y., Savchenko, A., Cort, J. R., Booth, V., Mackereth, C. D., Saridakis, V., Ekiel, I., Kozlov, G., Maxwell, K. L., Wu, N., McIntosh, L. P., Gehring, K., Kennedy, M. A., Davidson, A. R., Pai, E. F., Gerstein, M., Edwards, A. M. & Arrowsmith, C. H. (2000). Structural proteomics of an archaeon. Nat. Struct. Biol. 7, 903–909.
Cortes, D. M. & Perozo, E. (1997). Structural dynamics of the Streptomyces lividans K+ channel (SKC1): oligomeric stoichiometry and stability. Biochemistry, 36, 10343–10352.
Day, P. W., Rasmussen, S. G., Parnot, C., Fung, J. J., Masood, A., Kobilka, T. S., Yao, X. J., Choi, H. J., Weis, W. I., Rohrer, D. K. & Kobilka, B. K. (2007). A monoclonal antibody for G protein-coupled receptor crystallography. Nat. Methods, 4, 927–929.
Dobrovetsky, E., Lu, M. L., Andorn-Broza, R., Khutoreskaya, G., Bray, J. E., Savchenko, A., Arrowsmith, C. H., Edwards, A. M. & Koth, C. M. (2005). High-throughput production of prokaryotic membrane proteins. J. Struct. Funct. Genomics, 6, 33–50.
Dobrovetsky, E., Menendez, J., Edwards, A. M. & Koth, C. M. (2007). A robust purification strategy to accelerate membrane proteomics. Methods, 41, 381–387.
Doyle, D. A., Morais Cabral, J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T. & MacKinnon, R. (1998). The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science, 280, 69–77.
Engel, C. K., Chen, L. & Privé, G. G. (2002). Stability of the lactose permease in detergent solutions. Biochim. Biophys. Acta, 1564, 47–56.
Eshaghi, S., Hedrén, M., Nasser, M. I., Hammarberg, T., Thornell, A. & Nordlund, P. (2005). An efficient strategy for high-throughput expression screening of recombinant integral membrane proteins. Protein Sci. 14, 676–683.
Geertsma, E. R., Groeneveld, M., Slotboom, D. J. & Poolman, B. (2008). Quality control of overexpressed membrane proteins. Proc. Natl Acad. Sci. USA, 105, 5722–5727.
Gräslund, S., Nordlund, P., Weigelt, J., Hallberg, B. M., Bray, J., Gileadi, O., Knapp, S., Oppermann, U., Arrowsmith, C., Hui, R., Ming, J., dhe-Paganon, S., Park, H. W., Savchenko, A., Yee, A., Edwards, A., Vincentelli, R., Cambillau, C., Kim, R., Kim, S. H., Rao, Z., Shi, Y., Terwilliger, T. C., Kim, C. Y., Hung, L. W., Waldo, G. S., Peleg, Y., Albeck, S., Unger, T., Dym, O., Prilusky, J., Sussman, J. L., Stevens, R. C., Lesley, S. A., Wilson, I. A., Joachimiak, A., Collart, F., Dementieva, I., Donnelly, M. I., Eschenfeldt, W. H., Kim, Y., Stols, L., Wu, R., Zhou, M., Burley, S. K., Emtage, J. S., Sauder, J. M., Thompson, D., Bain, K., Luz, J., Gheyi, T., Zhang, F., Atwell, S., Almo, S. C., Bonanno, J. B., Fiser, A., Swaminathan, S., Studier, F. W., Chance, M. R., Sali, A., Acton, T. B., Xiao, R., Zhao, L., Ma, L. C., Hunt, J. F., Tong, L., Cunningham, K., Inouye, M., Anderson, S., Janjua, H., Shastry, R., Ho, C. K., Wang, D., Wang, H., Jiang, M., Montelione, G. T., Stuart, D. I., Owens, R. J., Daenke, S., Schütz, A., Heinemann, U., Yokoyama, S., Büssow, K. & Gunsalus, K. C. (2008). Protein production and purification. Nat. Methods, 5, 135–146.
Grisshammer, R. & Tate, C. G. (1995). Overexpression of integral membrane proteins for structural studies. Q. Rev. Biophys. 28, 315–422.
Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130.
Hanson, M. A., Cherezov, V., Griffith, M. T., Roth, C. B., Jaakola, V. P., Chien, E. Y., Velasquez, J., Kuhn, P. & Stevens, R. C. (2008). A specific cholesterol binding site is established by the 2.8 Å structure of the human beta2-adrenergic receptor. Structure, 16, 897–905.
Helenius, A., McCaslin, D. R., Fries, E. & Tanford, C. (1979). Properties of detergents. Methods Enzymol. 56, 734–749.
Hjelmeland, L. M. (1990). Removal of detergents from membrane proteins. Methods Enzymol. 182, 277–282.
Hunte, C. & Michel, H. (2002). Crystallisation of membrane proteins mediated by antibody fragments. Curr. Opin. Struct. Biol. 12, 503–508.
Ishihara, G., Goto, M., Saeki, M., Ito, K., Hori, T., Kigawa, T., Shirouzu, M. & Yokoyama, S. (2005). Expression of G protein coupled receptors in a cell-free translational system using detergents and thioredoxin-fusion vectors. Protein Expr. Purif. 41, 27–37.
Jaakola, V. P., Griffith, M. T., Hanson, M. A., Cherezov, V., Chien, E. Y., Lane, J. R., Ijzerman, A. P. & Stevens, R. C. (2008). The 2.6 Å crystal structure of a human A2A adenosine receptor bound to an antagonist. Science, 322, 1211–1217.
Klammt, C., Schwarz, D., Eifler, N., Engel, A., Piehler, J., Haase, W., Hahn, S., Dötsch, V. & Bernhard, F. (2007). Cell-free production of G protein-coupled receptors for functional and structural studies. J. Struct. Biol. 158, 482–493.
Krautwurst, D., Yau, K. W. & Reed, R. R. (1998). Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell, 95, 917–926.
Kuruma, Y., Nishiyama, K., Shimizu, Y., Müller, M. & Ueda, T. (2005). Development of a minimal cell-free translation system for the synthesis of presecretory and integral membrane proteins. Biotechnol. Prog. 21, 1243–1251.
Lemieux, M. J., Song, J., Kim, M. J., Huang, Y., Villa, A., Auer, M., Li, X. D. & Wang, D. N. (2003). Three-dimensional crystallization of the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamily. Protein Sci. 12, 2748–2756.
Lewinson, O., Lee, A. T. & Rees, D. C. (2008). The funnel approach to the precrystallization production of membrane proteins. J. Mol. Biol. 377, 62–73.
Lichty, J. J., Malecki, J. L., Agnew, H. D., Michelson-Horowitz, D. J. & Tan, S. (2005). Comparison of affinity tags for protein purification. Protein Expr. Purif. 41, 98–105.
Long, S. B., Campbell, E. B. & Mackinnon, R. (2005). Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science, 309, 897–903.
Long, S. B., Tao, X., Campbell, E. B. & MacKinnon, R. (2007). Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. Nature (London), 450, 376–382.
Lunin, V. V., Dobrovetsky, E., Khutoreskaya, G., Zhang, R., Joachimiak, A., Doyle, D. A., Bochkarev, A., Maguire, M. E., Edwards, A. M. & Koth, C. M. (2006). Crystal structure of the CorA Mg2+ transporter. Nature (London), 440, 833–837.
Magnani, F., Shibata, Y., Serrano-Vega, M. J. & Tate, C. G. (2008). Co-evolving stability and conformational homogeneity of the human adenosine A2a receptor. Proc. Natl Acad. Sci. USA, 105, 10744–10749.
Miroux, B. & Walker, J. E. (1996). Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298.
Neugebauer, J. M. (1990). Detergents: an overview. Methods Enzymol. 182, 239–253.
Newby, Z. E., O'Connell, J. D. III, Gruswitz, F., Hays, F. A., Harries, W. E., Harwood, I. M., Ho, J. D., Lee, J. K., Savage, D. F., Miercke, L. J. & Stroud, R. M. (2009). A general protocol for the crystallization of membrane proteins for X-ray structural investigation. Nat. Protoc. 4, 619–637.
Newstead, S., Ferrandon, S. & Iwata, S. (2008). Rationalizing alpha-helical membrane protein crystallization. Protein Sci. 17, 466–472.
Payandeh, J. & Pai, E. F. (2006). A structural basis for Mg2+ homeostasis and the CorA translocation cycle. EMBO J. 25, 3762–3773.
Prive, G. G. (2007). Detergents for the stabilization and crystallization of membrane proteins. Methods, 41, 388–397.
Raman, P., Cherezov, V. & Caffrey, M. (2006). The Membrane Protein Data Bank. Cell. Mol. Life Sci. 63, 36–51.
Rasmussen, S. G., Choi, H. J., Rosenbaum, D. M., Kobilka, T. S., Thian, F. S., Edwards, P. C., Burghammer, M., Ratnala, V. R., Sanishvili, R., Fischetti, R. F., Schertler, G. F., Weis, W. I. & Kobilka, B. K. (2007). Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature (London), 450, 383–387.
Rosenbaum, D. M., Cherezov, V., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., Choi, H. J., Yao, X. J., Weis, W. I., Stevens, R. C. & Kobilka, B. K. (2007). GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science, 318, 1266–1273.
Sakai, H. & Tsukihara, T. (1998). Structures of membrane proteins determined at atomic resolution. J. Biochem. 124, 1051–1059.
Savage, D. F., Anderson, C. L., Robles-Colmenares, Y., Newby, Z. E. & Stroud, R. M. (2007). Cell-free complements in vivo expression of the E. coli membrane proteome. Protein Sci. 16, 966–976.
Schwarz, D., Dötsch, V. & Bernhard, F. (2008). Production of membrane proteins using cell-free expression systems. Proteomics, 8, 3933–3946.
Schwarz, D., Klammt, C., Koglin, A., Lohr, F., Schneider, B., Dotsch, V. & Bernhard, F. (2007). Preparative scale cell-free expression systems: new tools for the large scale preparation of integral membrane proteins for functional and structural studies. Methods, 41, 355–369.
Serrano-Vega, M. J., Magnani, F., Shibata, Y. & Tate, C. G. (2008). Conformational thermostabilization of the beta1-adrenergic receptor in a detergent-resistant form. Proc. Natl Acad. Sci. USA, 105, 877–882.
Studier, F. W. & Moffatt, B. A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113–130.
Sukharev, S. I., Schroeder, M. J. & McCaslin, D. R. (1999). Stoichiometry of the large conductance bacterial mechanosensitive channel of E. coli. A biochemical study. J. Membr. Biol. 171, 183–193.
Surade, S., Klein, M., Stolt-Bergner, P. C., Muenke, C., Roy, A. & Michel, H. (2006). Comparative analysis and `expression space' coverage of the production of prokaryotic membrane proteins for structural genomics. Protein Sci. 15, 2178–2189.
Wagner, S., Klepsch, M. M., Schlegel, S., Appel, A., Draheim, R., Tarry, M., Högbom, M., van Wijk, K. J., Slotboom, D. J., Persson, J. O. & de Gier, J. W. (2008). Tuning Escherichia coli for membrane protein overexpression. Proc. Natl Acad. Sci. USA, 105, 14371–14376.
Wang, D. N., Safferling, M., Lemieux, M. J., Griffith, H., Chen, Y. & Li, X. D. (2003). Practical aspects of overexpressing bacterial secondary membrane transporters for structural studies. Biochim. Biophys. Acta, 1610, 23–36.
Warne, T., Serrano-Vega, M. J., Baker, J. G., Moukhametzianov, R., Edwards, P. C., Henderson, R., Leslie, A. G., Tate, C. G. & Schertler, G. F. (2008). Structure of a beta1-adrenergic G-protein-coupled receptor. Nature (London), 454, 486–491.
Warne, T., Serrano-Vega, M. J., Tate, C. G. & Schertler, G. F. (2009). Development and crystallization of a minimal thermostabilised G protein-coupled receptor. Protein Expr. Purif. 65, 204–213.
Wiener, M. C. (2004). A pedestrian guide to membrane protein cryst­allization. Methods, 34, 364–372.
Willis, M. S. & Koth, C. M. (2008). Structural proteomics of membrane proteins: a survey of published techniques and design of a rational high-throughput strategy. Methods Mol. Biol. 426, 277–295.

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