International
Tables for
Crystallography
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. 93-96

Section 3.2.3. A consensus strategy for membrane-protein purification

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:  koth.christopher@gene.com

3.2.3. A consensus strategy for membrane-protein purification

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3.2.3.1. 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. 3.2.3.1[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.

[Figure 3.2.3.1]

Figure 3.2.3.1 | top | pdf |

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.

3.2.3.2. 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.

3.2.3.3. 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.

3.2.3.4. 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.

3.2.3.5. 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]).

3.2.3.6. 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. 3.2.3.1[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]).

3.2.3.7. 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]).

References

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