[38] Purification of membrane proteins

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[38] P u r i f i c a t i o n o f M e m b r a n e P r o t e i n s

By

THOMAS C. THOMAS and MARK G. MCNAMEE

As our interest in molecular mechanisms responsible for cellular function has increased, the purification of membrane proteins has become an important part of many research efforts. The ultimate goal in purifying these proteins is to fully characterize them and determine how they fulfill their unique functional roles in oivo. Advances in our understanding of membrane proteins have recently culminated in the successful purification, crystallization, and X-ray crystallographic analysis of the photoreaction center from Rhodopseudomonas viridis.1 With continued advances in the field, it may soon be possible to fully characterize other multisubunit, integral membrane proteins, such as the nicotinic acetylcholine receptor and the voltage-dependent sodium channel. The methods available for the purification of membrane proteins are basically the same as those employed to purify water-soluble, nonmembrane-associated proteins. These methods include precipitation, gel filtration, ion-exchange, reversed-phase, and affinity chromatography (described in Section VII of this volume). Several unique characteristics of membrane proteins, however, often make it difficult to apply these methods successfully. In this chapter we will emphasize methods and conditions of purification which are designed to yield functionally active membrane proteins. Functional activity is judged by the ability of proteins to perform tasks such as catalyzing reactions, promoting ion flux, or binding specific ligands. This approach was emphasized in an excellent chapter in this series by Jos van Renswoude and Christoph Kempf. 2 We have attempted to extend the scope of this earlier chapter by emphasizing those areas where advances have been made, both in conventional chromatographic techniques and in newly developed genetic and immunological techniques. It is important to stress that, just as with soluble proteins, there is no way to present a single, precise set of methods for the purification of all membrane proteins. Each membrane protein possesses a unique set of physical characteristics, and conditions which are suitable for the purification of one protein may not be suitable for others. Table I lists a number t j. Deisenhofer, O. Epp, K. Miki, R. Huber, and H. Michel, Nature (London) 318, 618 (1985). 2 j. v a n R e n s w o u d e and C. K e m p f , this series, Vol. 104, p. 329.

METHODS IN ENZYMOLOGY, VOL. 182

Copyright © 1990by AcademicPress, Inc. All rights of reproduction in any form reserved.

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of well-characterized membrane proteins and the methods used to purify them. The first distinction which must be made is between peripheral and integral membrane proteins) Peripheral membrane proteins are predominantly hydrophilic in nature and are mainly associated with the membrane surface through electrostatic interactions. These proteins are easily solubilized and then purified by conventional chromatographic methods. Integral membrane proteins, however, are predominantly amphiphiles with hydrophilic regions exposed to the aqueous environment and hydrophobic regions embedded in the lipid matrix. These proteins must often be associated with lipids, which may differ in their charge and structure, in order to remain functionally active. 4 Prior to purification, these lipid-associated integral membrane proteins must be extracted from the membrane. This solubilization is typically achieved with amphiphilic detergents, which may also differ in their net charge and structure. As a result, the solubilization of a membrane protein, under conditions that preserve its functional activity, yields a very heterogeneous detergent-lipid-protein micelle. Attempts to chromatographically purify the protein may change the composition of this assembly, thereby inactivating or altering the properties of the protein. Chromatographic methods which provide the highest yield of active, functional protein are those that alter this soluble assembly the least. As a result, the dual goals of preserving functional activity and of attaining high chromatographic resolution are often diametrically opposed.

Preparation of Membranes Preparation of a suitable membrane fraction is the first stage in the process of isolating and purifying a membrane protein. The methods currently available for isolating enriched membranes are typically of low resolution (2- to 5-fold increase in specific activity), but yields are generally high and significant amounts of contaminating material can be removed. Membrane proteins are most stable while they are embedded in the membrane, and time invested at this stage will improve results during subsequent stages of the purification. The ability to subfractionate membranes is the only way in which membrane proteins offer unique advantages over soluble proteins. The first step is to obtain a tissue in which the protein of interest has a high specific activity, An excellent example is the electric organ of Tor3 S. J. Singer and G. L. Nicolson, Science 175, 720 (1972). 4 0 . T. Jones, J. H. Eubanks, J. P. Earnest, and M. G. McNamee, Biochemistry 27, 3733 (1988).

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pedo californica, from which the nicotinic acetylcholine receptor (AchR) has been purified. Due to the high density of receptor-rich synapses in this tissue, only a 50-fold purification is necessary in order to obtain pure AchR. 5 Another example is the human erythrocyte membrane, from which plasma membrane proteins are easily obtained without contamination by other cell types or by intracellular membrane proteins. 6 While the choice of a tissue or cell source is an important first step in the purification of both soluble and membrane proteins, it may be especially important for the purification of membrane proteins. There are very few examples in which membrane proteins of low abundance have been successfully purified. The next step generally entails subcellular fractionation. Many diverse methods have been developed, and applications involving both animal and plant cells can be found in two recent reviews 7,8 (see also [16] in this volume). The most frequently used methods employ a combination of differential centrifugation and sucrose density gradient centrifugation steps. More recently, Percoll has been substituted for sucrose in order to form density gradients which are nearly isosmotic. The best fractionation method to use for enrichment of a particular protein is determined on the basis of a careful balance sheet of yields, changes in specific activity, and the distributions of marker enzymes. Separations using differential centrifugation are rapid and recoveries are high. Sucrose gradients offer improved purification, but they are more time consuming. If the protein of interest is distributed among several gradient fractions then very little improvement in specific activity is seen and yields are low. In these cases, it is common to prepare a crude membrane fraction and use this as the initial source of protein for solubilization. One rapid, high-yield method involves homogenizing the tissue in 10 vol of a 0.25 M sucrose buffer at 4°, followed by centrifugation at 1000 g for 10 min. The supernatant fraction is then centrifuged at 105,000 g for 1 hr at 4° and the crude membrane pellet is recovered. 9 This method yields membranes which are free of whole cells, nuclei, and soluble proteins. During cell disruption harmful proteases may be released. Compounds which have been very effective at minimizing proteolysis of membrane proteins include EDTA and EGTA (0.1-5 mM), which inhibit divalent cation-dependent proteases; phenylmethylsulfonyl fluoride (PMSF, 0.1-1 5 R. L. Vandlen, W. C.-S Wu, J. C. Eisenach, and M. A. Raftery, Biochemistry 18, 1845 (1979). 6 G. Fairbanks, T. L. Steck, and D. F. H. Wallach, Biochemistry 10, 2606 (1971). 7 W. H. Evans, in "Biological Membranes: A Practical Approach" (J. B. C. Findlay and W. H. Evans, eds.), p. 1. IRL Press, Oxford, 1987. 8 j. D. Morre, A. O. Brightman, and A. S. Sandelius, see ref. 7, p. 37. 9 j. Ramwani and R. K. Mishra, J. Biol. Chem. 261, 8894 (1986).

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mM), which inhibits serine proteases; and N-ethylmaleimide (0.1-1 mM), which inhibits sulfhydryl proteases (see Ref. 7 for additional protease inhibitors). PMSF has a short half-life in aqueous solution and is prepared as a stock solution (100 mM) in ethanol or 2-propanol. Dithiothreitol (DTT, 0.1-1 mM) is often added to prevent oxidation. Since most of these compounds have the potential to covalently modify and/or alter the activity of membrane proteins, caution is advised when using them.

Purification of Peripheral Membrane Proteins When preparing a membrane fraction for the purification of peripheral membrane proteins, several additional factors will influence the choice of methods. Membranes intended for this purpose should be prepared under isosmotic conditions (e.g., 0.15 M NaCI). High ionic strength solutions may result in the premature solubilization of peripheral membrane proteins, and low ionic strength buffers may result in nonspecific association of soluble proteins with the membrane. In addition, some proteins exist in both soluble and membrane-associated forms in v i v o . Jo These proteins can bind reversibly to either the protein or lipid portions of the membrane. Reversible binding may involve conformational changes in the protein induced by changes in cation concentrations or phosphorylation. One example is Ca2+/phospholipid-dependent protein kinase, which binds to the membrane upon activation in a Ca2+-dependent manner.~l Therefore, the composition of the buffer used during the preparation of a membrane fraction may affect the distribution of these proteins between the soluble and membrane-associated fractions. Peripheral membrane proteins are solubilized by incubating membranes with solutions which interrupt electrostatic and in some cases hydrophobic interactions. Solutions containing one or more of the following compounds are commonly used for this purpose. 1. NaCI or KCI (>0.15 M) 12 2. Buffers of acidic (3-5) or basic (8-12) pH j3 3. EDTA and EGTA: These are frequently added to destabilize bonds that are enhanced by Mg 2+ and C a 2+ 14 4. Chaotropic agents (containing I-, Br-, CIO4-, and SCN- ions)iS: l0 p. Burn, TIBS 13, 79 (1988). i1 j. H. Schwartz, and S. M. Greenberg, Annu. Reo. Neurosci. 10, 459 (1987). 12 H. W. Chang and E. Bock, Biochemistry 16, 4513 (1977). t3 T. Yoshihisa, Y. Ohsumi, and Y. Anraku, J. Biol. Chem. 263, 5158 (1988). ~4 B. J. Bowman, F. Blasco, and C. W. Slayman, J. Biol. Chem. 256, 12343 (1981). ~5V. Bennett, K. Gardner, and J. P. Steiner, J. Biol. Chem. 263, 5860 (1988).

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These agents act by disordering the structure of water. This tends to reduce the strength of hydrophobic bonds, thereby increasing the solubility of partially hydrophobic proteins in the aqueous phase 5. Strong denaturing agents, such as urea and guanidine hydrochloride 16 6. Diiodosalicylate or sulfosalicylatel7: These salts may have detergent-like characteristics 7. Detergents, such as deoxycholate or Triton X-100 (discussed later). 14,18 Integral membrane proteins will also be solubilized by these treatments. After incubation for 10 to 60 min on ice, insoluble membranes are removed by centrifugation at 100,000 g for 60 min. The efficiency of solubilization should be monitored by performing activity and protein assays to determine the distribution and specific activity of desired proteins. The distribution of proteins may also be monitored by SDS-PAGE. The solubilized peripheral membrane proteins are then fractionated by methods similar to those applied to soluble proteins. In some cases it may be necessary to include salts and detergents in buffers during chromatography in order to prevent the aggregation and precipitation of proteins.~5 Care must be taken when using these solutions since they all have the ability to inactivate or denature membrane proteins. Solubilization of membrane proteins by one of the first four solutions listed above is commonly used as a method to distinguish between peripheral and integral membrane proteins. In fact, membranes are frequently preextracted with these solutions to remove peripheral membrane proteins prior to the solubilization of integral membrane proteins by detergents. Treatment of membranes with high enough concentrations of these solutions can, however, result in the solubilization of some integral membrane proteins. In one example, a lipid-associated protein was extracted from presynaptic membranes by alkaline extraction.~9 Solubilization of Integral Membrane Proteins In order to purify integral membrane proteins by chromatographic methods, the proteins must be removed from the lipid bilayer and individually dispersed into solution. This is most effectively accomplished with ~6N. J. Newman, D. L. Foster, T. H. Wilson, and H. R. Kaback, J. Biol. Chem. 256, 11804 (1981). 17 j, K. Wright and P. Overath, Eur. J. Biochem. 138, 497 (1984), t8 W. J. LaRochelle and S. C. Froehner, J. Biol. Chem. 262, 8190 (1987). t9 M. Israel, N. Morel, B. Lesbats, S. Birman, and R. Manaranche, Proc. Natl. Acad. Sci. U.S.A. 83, 9226 (1986).

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amphiphilic detergents in aqueous solution, although in a few cases organic solvents have been used. Chapters [18] and [19] of this volume contain a very thorough discussion of the different types of detergents and the best methods for using them. We will, therefore, provide only a brief overview of detergents and their use as it applies to chromatography of proteins. Detergents may be grouped into two classes, ionic and nonionic. The ionic detergents are either anionic (e.g., sodium dodecyl sulfate and the bile salts, cholate and deoxycholate), cationic (e.g., alkyltrimethylammonium salts), or zwitterionic (e.g., Zwittergent and CHAPS). These detergents are generally very effective at solubilizing integral membrane proteins and dissociating protein complexes. Some ionic detergents (e.g., sodium dodecyl sulfate) are very denaturing and are used only when recovery of functional activity is unimportant. Ionic detergents generally have a high (> 1 mM) critical micelle concentration (cmc) and are easily removed by methods such as dialysis. Cholate, deoxycholate, and CHAPS form small micelles (4-6 kDa) which do not interfere with gel filtration. Due to their charge, however, cholate and deoxycholate are not suitable for ion-exchange chromatography. Examples of nonionic detergents include octylglucoside, digitonin, and the polyoxyethylene derivatives (e.g., Triton X-100, Lubrol PX, and the Tween series). These detergents are less effective at dissociating protein complexes, but many proteins are more stable in nonionic detergents than in ionic detergents. Nonionic detergents generally have a low cmc (< 1 mM) and are difficult to remove without using special resins such as Bio-Rad SM-2 beads. 2° In addition, the polyoxyethylene derivatives and digitonin form large micelles which may interfere with gel filtration. Triton X-100 absorbs at 280 nm and interferes with the use of ultraviolet absorbance methods to monitor the chromatographic elution of proteins. Octylglucoside is an important exception to the above statements about nonionic detergents. It has a high cmc (23.3 mM), forms small micelles (8 kDa), and does not absorb light at 280 nm. At the present time no detergent has emerged as the best choice for solubilizing a broad range of proteins, but on a practical basis we would suggest screening CHAPS and octylglucoside first. Detergents are screened by preparing membrane fractions at a specific protein concentration and adding extraction solutions with a range of detergent concentrations. Greatest success is achieved when using final protein concentrations of 1 to 10 mg/ml and detergent/protein ratios of 0.1 to 10 (w/w). Solutions are incubated at 0-4 ° for 30 to 60 min (longer incubations may be necessary) and then centrifuged at 105,000 g for 1 hr 20

p. W. Holloway, Anal. Biochem. 53, 304 (1973).

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at 4°. 2~ By definition, solubilized proteins remain in the supernatant solution. Both the specific activity and the yield of soluble, functionally active protein should be determined. The optimum detergent/protein ratio for solubilization will in most cases be chosen as a compromise between yield and specific activity. Detergents are generally most effective at concentrations above their cmc. In some cases aggregates or assemblies of proteins which have not been fully dissociated are found to be soluble by the above criterion. This is most likely to happen when nonionic detergents are used or when glycerol, sucrose, or urea are added to extraction solutions. These additives will increase the solution density with the effect that some membrane fragments may not sediment. Another method which can be used to determine solubility is gel filtration chromatography. Proteins which elute after the void volume are considered soluble. 22 Using this method, it was determined that CHAPS is far more effective at solubilizing erythrocyte membrane proteins than reduced Triton X-100 (reduction eliminates absorbance at 280 nm) or Tween 20. 2zJust as with sedimentation by centrifugation, this definition of solubility is operational and will depend on the fractionation range of the gel used. In some cases it has been found that a mixture of detergents will provide the most effective solubilization of active pr0tein. 23 Also, the yield of protein solubilized in an active state can sometimes be improved by including lipid in the solubilization b u f f e r . 24 It is not clear whether the addition of exogenous lipid protects the protein during extraction, thereby allowing more complete solubilization at higher detergent concentrations, or stabilizes the protein after solubilization. In most cases, however, the addition of lipid will simply lower the effective concentration of the detergent. Higher concentrations of detergent will then be needed to achieve the same yield. Organic solvents may sometimes be used as an alternative to detergents. ~9 These solvents denature most membrane proteins and only a small percentage of proteins will be soluble in the organic phase. An additional consideration is that once solubilized in organic solvent, it may be difficult to determine the activity or function of proteins in an aqueous assay system. Those solvent systems which have been used with greatest success have been well described elsewhere. 2 21 L. M. Hjelmeland and A. Chrambach, in "Membranes, Detergents, and Receptor Solubilization" (J. C. Venter and L. C. Harrison, eds.), p. 35. Alan R. Liss, New York, New York, 1984. 22 R. S. Matson and S. C. Goheen, J. Chromatogr, 359, 285 (1986). 23 C. R. Cremo, G. S. Hen'on, and M. I. Schimerlik, Anal. Biochem. 115, 331 (1981). z4 R. P. Hartshorne and W. A. Catterall, J. Biol. Chem. 259, 1667 (1984).

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Fractionation of Integral Membrane Proteins Once a suitable preparation of soluble membrane proteins has been prepared, fractionation methods can be employed to isolate a specific protein of interest. These methods include conventional chromatographic techniques (Section VII, this volume) and a few methods specific for membrane proteins. The most widely used chromatographic techniques are gel filtration, affinity, ion-exchange, and reversed-phase chromatography. The following section describes methods for optimizing the use of these techniques with integral membrane proteins. Gel Filtration

This technique is useful for bulk separation of high-, medium-, and low-molecular-weight membrane proteins. High yields of active protein are frequently achieved, but the usefulness of this technique is limited by the low levels of purification which are typically obtained ( < 5 - f o l d ) . 25'26 The conditions chosen to solubilize active protein will yield a mixture of detergent-protein micelles, detergent-lipid-protein micelles, and possibly small membrane fragments. 27 This heterogeneity can result in a broad distribution for each specific protein. In order to prevent detergent-mediated inactivation of proteins during gel filtration, it is common practice to use detergent in the eluant buffer at a concentration 10- to 100-fold lower than used during the solubilization step. 25 This practice can promote nonspecific aggregation and further decrease the level of purification which is attained. 28 In addition, if exogenous lipid has been added to the elution buffer, proteins may be partially reconstituted into vesicles and elute in the void volume. There are several steps which may be taken to increase resolution and yield, and to prevent protein aggregation. 1. Use large-pore chromatography resins such as Sephacryl S-300 and S-400 (Pharmacia). Detergent-protein complexes may exhibit twice the apparent molecular weight expected for the protein alone, and nondenaturing detergents such as Triton X-100 may not fully dissociate protein complexes or aggregates, z2 As a result it is common for detergent-solubilized protein complexes to elute with apparent molecular weights between 200,000 and 1,000,000. When using gel filtration resins designed for HPLC 2~ A. F. Weiton, P. M. Lad, A. C. Newby, H. Yamamura, S. Nicosia, and M. Rodbell, Biochim. Biophys. Acta 522, 625 (1978). 26 E. C. Hulme, C. P. Berrie, T. Haga, N. J. M. Birdsall, A. S. V. Burgen, and J. Stockton, J. Recept. Res. 3~ 301 (1983). z7 A. Helenius and K. Simons, Biochim. Biophys. Acta 415, 29 (1975). 28 A. C. Newby and A. Chrambach, Biochem. J. 177, 623 (1979).

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and FPLC systems, the best recoveries will be achieved with larger particle sizes. 29 2. Gel filtration protocols should be optimized by determining the resolution and recovery of functionally active proteins at several detergent concentrations. In most cases there will be a minimum detergent concentration which is necessary in the elution buffer in order to achieve maximum resolution. Bacteriorhodopsin from Halobacterium required 2.0% (w/w) Triton X-100 (ca. 10× cmc) in both the extraction and elution buffers, and 6 hr of incubation, before it was completely dissociated into monomers. 3° Analysis of rat liver microsomal proteins determined that the minimum concentration of octaethylene glycol dodecyl ether (Ci2E8) which was required for optimal resolution was 0.05% (w/w) (10× cmc). 31 These studies clearly indicate that the practice of reducing detergent concentrations in the elution buffer can reduce the level of resolution and purification obtained. The optimal level of detergent in the elution buffer (always > cmc) will be chosen as a compromise between maximal resolution and high recovery of active protein. It should be noted that elution conditions can be rapidly varied and tested on HPLC or FPLC systems. The use of these systems should greatly expand the application of gel filtration to membrane protein purification. 3. Examine the use of detergent mixtures. When adenylate cyclase was solubilized in 1% Lubrol PX (nonionic) and chromatographed in 0.01% Lubrol PX, aggregation occurred. Solubilization of adenylate cyclase in deoxycholate inactivated the enzyme. However, addition of deoxycholate to the Lubrol PX-containing elution buffer (1 : 3, w/w) prevented aggregation while maintaining 90% of the activity. 28 Gel filtration should be employed as the first step in the purification protocol for two reasons. First, most methods for concentrating membrane proteins are very poor (discussed later), resulting in low yields and aggregation. Since gel filtration requires a concentrated sample of relatively small volume, it is best to extract membranes at a high protein concentration (5-l0 mg/ml) and immediately chromatograph by gel filtration. The dilute sample which is isolated by this procedure can then be purified by either ion-exchange or affinity chromatography since these are suitable methods for use with dilute samples. The second reason is that gel filtration can be used to determine whether or not the protein of interest has been completely solubilized. Optimal solubilization conditions should produce a symmetrical peak of activity which is found com29 G. W. Welling, K. Slopsema, and S. Welling-Wester, J. Chromatogr. 359, 307 (1986). 3o R. Pabst, T. Nawroth, and K. Dose, J. Chromatogr. 285, 333 (1984). 31 y . Kato, T. Kitamura, K. Nakamura, A. Mitsui, Y. Yamasaki, and T. Hashimoto, J. Chromatogr. 391, 395 (1987).

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pletely within the included volume. If the protein has not been solubilized in a uniform manner, the level of purification attained during subsequent purification steps will also be reduced.

Affinity Chromatography This is by far the most useful and successfully applied method for purification of integral membrane proteins. In fact, it has played an essential role in the purification of large multisubunit receptors. Although nonaffinity methods generally provide less than a 20-fold purification of membrane proteins, affinity chromatography routinely achieves purifications of between I000-and 10,000-fold. Three types of affinity chromatography will be described. These include the use of specific ligands (e.g., enzyme inhibitors, hormones, neurotransmitters), a general ligand (lectins), and antibodies. Specific affinity ligands immobilize the protein of interest without disturbing the interactions among protein, detergent, and lipid. The immobilized protein can then be eluted by equally gentle methods using a soluble ligand. If the solubilized protein is active in detergent, then it is not necessary to remove the detergent prior to the adsorption step. When a specific ligand is used the major obstacles to attaining a high degree of purification are protein aggregation and nonspecific binding to the column. A level of detergent sufficient to prevent protein aggregation while not inactivating the protein of interest should be included in the buffer. A high ionic strength buffer will reduce nonspecific binding due to electrostatic interactions, and the presence of detergent in the buffer should reduce nonspecific hydrophobic interactions. It is important to note that changing the ionic strength of the buffer, in order to weaken nonspecific interactions, may also affect the strength of specific ligand binding. In some cases the combination of specific and nonspecific interactions may require the simultaneous use of a strong dissociating agent, such as urea, and a soluble ligand in order to elute the protein of interest. 3~a In addition it is recommended that ligands be attached to the column resin by a hydrophilic (rather than hydrophobic) spacer arm. 32,33 This will increase both the specificity of the binding and the yield of protein. The level of purification achieved may also be improved by using a low ligand density, of by using 31a G. B. Stauber, R. W. Ransom, A. I. Dilber, and R. W. Olsen, Eur. J. Biochem. 167, 125 (1987). 32 G. Vauquelin, P. Geynet, J. Hanoune, and A. D. Strosberg, Eur. J. Biochem. 98, 543 (1979). 33 E. Sigel, A. Stephenson, C. Mamalaki, and E. A. Barnard, J. Biol. Chem. 258, 6965 (1983).

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a minimum amount of the affinity resin. 33'34 Methods for preparing and analyzing affinity resins are discussed elsewhere (see [29] in this volume; see also Ref. 35). Although the binding of protein to the affinity ligand should not disturb the interactions among detergent, protein, and lipid, the immobilization of these micelles may create additional problems. Since only the protein portion directly binds to the affinity resin, detergent-containing buffers may extract and elute the lipid portion of these bound complexes. This may inactivate and possibly even elute the protein. For this reason, lipids are frequently included in elution buffers (1-2%, w/v) in order to stabilize active proteins. 4 Crude lipid mixtures (e.g., soybean asolectin and bovine brain total lipid extract) or purified lipids (e.g., phosphatidylcholines) may be used. A more general form of affinity chromatography utilizes lectins as the immobilized ligand. Lectins are carbohydrate-binding proteins of nonimmune origin which offer a rapid and mild method to purify plasma membrane glycoproteins. Lectin-glycoprotein interactions are reversible and can be inhibited by simple sugars. Therefore, adsorbed proteins can be eluted from lectin columns using simple sugars without changes in pH and ionic strength and without the use of denaturants. However, this method is unable to achieve significant subfractionation of glycoproteins and therefore yields lower levels of purification than achieved with more specific types of affinity chromatography. Hydrophobic and ion-exchange effects may also cause nonspecific binding. In addition, lectins are very sensitive to treatment with certain types of detergents. Nonionic detergents (up to 2.5%, w/v) have negligible effects on lectin affinity, but ionic detergents such as deoxycholate and SDS significantly reduce the binding capacity of the most commonly used lectins. 36 The saccharides most commonly found attached to animal cell glycoproteins are sialic acid, galactose, mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. Numerous lectins have been identified which bind to each of these. 37 The most widely used lectins are concanavalin A (binds ~-D-mannose) and wheat germ agglutinin [binds sialic acid and (fl-D-GIcNAc)n]. 37'38 Although different lectins may have 34 S. A. Spencer, R. G. Hammonds, W. J. Henzel, H. Rodriguez, M. J. Waters, and W. I. Wood, J. Biol. Chem. 263, 7862 (1988). 35 C. R. Lowe, "Laboratory Techniques in Biochemistry and Molecular Biology" (T. S. Work and E. Work, eds.), Vol. 7, Part 2. North-Holland, Amsterdam, 1979. 36 R. Lotan, G. Beattie, W. Hubbell, and G. L. Nicolson, Biochemistry 16, 1787 (1977). 37 I. J. Goldstein and C. E. Hayes, Adv. Carbohydr. Chem. Biochem. 35, 127 (1978). 38 M. Monsigny, A.-C. Roche, C. Sene, R. Maget-Dana, and F. Delmotte, Eur. J. Biochem. 104, 147 (1980).

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the same specificity for sugars, their affinities will vary. The binding capacity of lectins for a specific protein can be assessed by determining their ability to precipitate that protein) 9 Suitable affinity resins can then be obtained commercially or prepared by the cyanogen bromide method.35:0 Some lectins, such as concanavalin A, and lentil and pea lectins require Ca 2÷ and Mn 2÷ (1 mM) for carbohydrate binding. This will affect the choice of buffers and whether or not chelating agents can be used. Adsorption is best performed in 0.15 M buffers of near neutral pH. After the column is washed, proteins are eluted with the same buffer containing an appropriate sugar (0.1-0.5 M). Gradient elution generally does not offer any advantages over step elution. Immunoaffinity ligands (antibodies) have been used with some success in the purification of membrane proteins, especially those of cell surface and viral origin. The major limitation to this technique is the strength with which antibodies bind their target proteins. Most methods of elution are very harsh and proteins isolated by this technique are generally inactive. Monoclonal or polyclonal antibodies have both been used. 4~,42Antibodies may be linked to CNBr-activated Sepharose (Ref. 40, also commercially available). In some cases, it may be necessary to incubate proteins with the immunoaffinity resin for extended periods of time. 41 Nonbound and nonspecifically bound proteins are then preeluted using neutral buffers containing moderately high levels of salt (<0.5 M NaCI) and detergent. Some commonly used elution buffers are as follows: 1. High or low pH buffer: For example, 0.05 M diethylamine (pH 11.5) or 50 mM citrate (pH 3 . 0 ) . 43'43a Eluted proteins are quickly neutralized to minimize loss of functional activity 2. Chaotropic agents, such as 3 M potassium thiocyanate 4~ Other methods of elution include the use of high salt concentrations and denaturants such as SDS, urea, and guanidine-HCl. In one case, polyclonal antibodies were produced against a synthetic peptide. The bound protein was eluted with excess peptide and mild buffer conditions which resulted in a 2500-fold purification. 42 39 F. Rieger and M. Vigny, J. Neurochem. 27, 121 (1976). 4o S. C. March, I. Parikh, and P. Cuatrecasas, Anal. Biochem. 60, 149 (1974). 41 j. j. Hubert, D. B. Schenk, H. Skelly, and H. L. Leffert, Biochemistry 25, 4156 (1986). 4z G. Walter, M. A. Hutchinson, T. Hunter, and W. Eckhart, Proc. Natl. Acad. Sci. U.S.A. 79, 4025 (1982). 43 I. S. Trowbridge and M. B. Omary, Proc. Natl. Acad. Sci. U.S.A. 78, 3039 (1981). 43a p. j. Whiting and J. M. Lindstrom, Biochemistry 25, 2082 (1986).

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Ion Exchange Purifications on the order of 5-fold are typical in successful applications of this technique. 24,44High yields of active protein (>80%) have been reported using this method, but there are many cases in which complete loss of activity has also occurred. This has led to the suggestion that some membrane proteins may be irreversibly bound by ion-exchange resins. It is more likely, however, that these proteins are inactivated. Ion-exchange resins not only immobilize proteins, but they also bind charged detergents and lipids. Ion-exchange chromatography, therefore, has the potential to pull apart detergent-lipid-protein complexes. As a first step, only neutral or zwitterionic detergents should be employed. The addition of defined mixtures of neutral and zwitterionic lipids to buffers may also stabilize proteins. In cases of low yields, minimal washing to remove unbound proteins followed by step elution rather than gradient elution may also improve results. 24,45 Glycerol (10-20%) is often used to minimize hydrophobic interactions with the resin and to stabilize proteins. 44,46 Work with rat microsomal membrane proteins indicates that detergent concentrations must be above a certain level, which depends on the detergent, in order to prevent poor resolution and recovery. 3~ Some proteins have specific requirements for charged lipids and cofactors which may be removed from the protein by attachment to the ion-exchange resin or by extraction with the elution buffer. 47 It may not be possible to recover these proteins in an active state. In cases where high yields of inactive protein are obtained, it may be possible to reactivate these proteins by adding back required factors. For example, delipidated Na+,K+-ATPase can be reactivated by addition of negatively charged phosphatidylserine. 47 Crude lipid mixtures such as asolectin may also be useful.

Reversed Phase Reversed-phase chromatography on HPLC columns can achieve highresolution separation of many proteins. Unfortunately, the resins and the organic solvents used with this technique denature most proteins. For this reason, reversed-phase chromatography is used predominantly as an analytical rather than a preparative method for purifying membrane proteins. Highly hydrophobic membrane proteins bind very tightly, and they are

44 N. Muto and L. Tan, J. Chromatogr. 326, 137 (1985). 45 W.-M. Hou, Z.-L. Zhang, and H.-H. Tai, Biochim. Biophys. Acta 959, 67 (1988). 46 G. W. Wellig, R. van der Zee, and S. Welling-Wester, J. Chromatogr. 418, 223 (1987). 47 M. I. DeCaldentey and K. P. Wheeler, Biochem. J. 177, 265 (1979).

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recovered in low yields when methods which were developed for use with soluble proteins are employed. In addition, detergents used to solubilize membrane proteins will also bind to reversed-phase resins. Therefore, modified methods have been developed for the successful application of reversed-phase chromatography to the analysis of membrane proteins. Membrane proteins can be directly solubilized into concentrated solutions of trifluoroacetic acid or formic acid, thus alleviating problems associated with the use of detergents. 48'49 Samples must be applied directly to columns, since prolonged treatment can lead to esterification of serine and threonine residues, deamination of glutamine and asparagine, and cleavage of Asp-Pro bonds. An alternative is to solubilize proteins with detergent, and then remove the detergent and lipid by chloroform-methanol extraction. 5° Proteins are removed from the water-solvent interface or recovered by centrifugation and then solubilized in acid as above. In some cases, detergent solubilized samples have been applied directly to columns and then eluted with organic solvents. 5~ Once bound to the column, proteins are typically eluted with a gradient of acetonitrile or 2propanol. Improved resolution and yield can be obtained by using columns developed for hydrophobic interaction chromatography rather than reversed-phase chromatography. 44,48,5°,51 These contain hydrophobic ligands of shorter chain length or lower'hydrophobicity such as C3, Ca, or phenyl groups. In addition some of these columns have a lower ligand density and a secondary hydrophilic phase (e.g., Bio-Rad BioGel TSK Phenyl-5PW). Proteins can be eluted at lower solvent concentrations if less polar solvents such as 2-propanol are used instead of methanol or acetonitrile. 46 In some cases the use of solvent mixtures such as acetonitrile-propanol have increased resolution. 52 Use of poor ion-pairing agents, such as phosphoric or hydrochloric acid rather than trifluoroacetic acid, will also reduce the strength of hydrophobic binding. 46 In addition, when HPLC-suitable columns are used, yields are better with large-pore columns (100 nm). 5~ In general, steps to reduce the amount of time in which proteins remain bound to the column will increase yields.

Miscellaneous Chromatographic Methods Other methods include adsorption on hydroxylapatite,45 chromatofocusing, 53 isoelectric focusing, z6 and sucrose or glycerol density gradi48 M. R. Sussman, Anal. Biochem. 169, 395 (1988). 49 j. Heukeshoven and R. Dernick, J. Chromatogr. 326, 91 (1985). 5o K. R. Brunden, C. T. Berg, and J. F. Poduslo, Anal. Biochem. 164, 474 (1987). 5~ S. C. Goheen and T. M. Chow, J. Chromatogr. 359~ 297 (1986). 52 G. E. Tarr and J. W. Crabb, Anal. Biochem. 131, 99 (1983). 33 B. R. Aton, B. J. Litman, and M. L. Jackson, Biochemistry 23, 1737 (1984).

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e n t s . 14,24 The principles involved in attaining high resolution and high

yields of functional membrane proteins with these methods are the same as those discussed above.

Phase Separation Elevated temperatures tend to reduce the solubility of detergents and induce phase separation. The temperature at which phase separation occurs is a characteristic of the detergent used (Triton X-100, 64°; Triton X-114, 200). 54 When phase separation occurs, hydrophilic proteins partition into the aqueous phase and hydrophobic proteins partition into the detergent phase. Therefore, phase separation after solubilization of proteins with Triton X-114 has been proposed as a practical way to separate peripheral from integral membrane proteins. Experience has shown, however, that many proteins do not follow this rule. For example, integral membrane proteins with very large hydrophilic regions, such as the nicotinic acetylcholine receptor (AchR) and the a subunit of the Na + channel, partition into the aqueous phase. 55 Phase separation of other detergents (e.g., Triton X-100 and cholate) can be induced at nondenaturing temperatures by the addition of ammonium sulfate. 56 It is important to note that these methods cause a substantial alteration in the composition of the solution and many proteins may not be stable under these conditions.

Transport Specificity Fractionation Goldin and colleagues have used the calcium oxalate density perturbation method to isolate the synaptosomal ATP-dependent C a 2+ uptake system. 57 Solubilized proteins were reconstituted into oxalate-containing artificial vesicles using an 80-fold excess of exogenous lipid. Under these conditions, each vesicle was expected to contain one protein (or at most a few). The addition of ATP induced the vesicles to take up Ca z+. Calcium oxalate formed and precipitated within the vesicles, thereby greatly enhancing their density. These vesicles were then purified 100-fold by density gradient centrifugation. Although this technique takes advantage of the specific transport properties of ion channels, the number of successful applications is limited. 54 C. Bordier, J. Biol. Chem. 256, 1604 (1981). 55 B. F. X. Reber and W. A. Catterall, J. Biol. Chem. 262, 11369 (1987). 56 C. R. Parish, B. J. Classon, J. Tsagaratos, I. D. Walker, L. Kirszbaum, and I. F. C. McKenzie, Anal. Biochem. 156, 495 (1986). _v D. Papazian, H. Rahamimoff, and S. M. Goldin, Proc. Natl. Acad. Sci. U.S.A. 76, 3708 (1979).

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Reconstitution of Integral Membrane Proteins Reconstitution of a functionally active membrane protein involves the incorporation of the solubilized membrane protein into artificial membranes with preservation of the original function of the protein. Many proteins are only functionally active when correctly positioned in a lipid bilayer. The ion flux through channels such as AchR and the Na + channel can only be measured when these proteins are reconstituted into lipid vesicles of an appropriate composition. Reconstitution is therefore an integral part of many purification schemes. Generally, reconstitution involves the addition of detergent-solubilized lipids to a detergent-solubilized protein sample, Subsequent removal of the detergent results in the formation of lipid bilayer structures and vesicles. It is therefore important that a detergent which is easily removed during reconstitution be used (e.g., cholate, deoxycholate, and octylglucoside). The most common method of reconstitution involves removing detergent by dialysis. 57 Other methods for detergent removal include gel filtration, preferential adsorption, and dilution.16.17 See E. Racker for a full description of reconstitution methods. 58

Renaturation of Functionally Active Proteins after Purification The premise of most of the techniques described so far has been that activity must be maintained at each step in the purification. Nevertheless, solubilization conditions which maintain activity often reduce chromatographic resolution. Mild nonionic detergents such as Triton X-100 often fail to prevent aggregation or to dissociate many protein complexes. On the other hand, harsh ionic detergents such as sodium dodecyl sulfate (SDS) thoroughly disperse proteins into monomers, but cause a loss of functional activity. Hjerten et al. have proposed a different approach to purification of membrane proteins. 59 They suggest that proteins should be solubilized and purified in SDS. After purification proteins can then be renatured by removal of SDS. Several membrane-associated proteins, including a 5'-nucleotidase from Acholeplasma laidlawii and a neuramidase from influenza virus, have been successfully purified in SDS and renatured by dialysis against heptaoxyethylene lauryl ether (a neutral detergent). There is additional evidence to support this approach. For example, bacteriorhodopsin solubilized in SDS still possessed approximately 50% 5s E. Racker, this series, Vol. 55, p. 699. 59 S. Hjerten, M. Sparrman, and J.-L. Liao, Biochim. Biophys. Acta 939, 476 (1988).

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of its a-helical structure.6° Even when bacteriorhodopsin was completely denatured with formic acid-ethanol, it could be renatured by addition of phospholipids, cholate, and retinal. Removal of the cholate by dialysis resulted in functionally reconstituted protein. Whether the purification of integral membrane proteins under denaturing conditions followed by functional reactivation will become a generally applicable method remains to be seen. It may be that this method is limited to relatively small, single-subunit proteins.

Genetic and Immunological Methods The past decade has seen an explosion in the development of new genetic and immunological techniques. Many of these have direct application to the identification and purification of membrane proteins. The advent of techniques to isolate and sequence full-length cDNA clones of genes has made possible the comparison of primary sequences from important membrane proteins. It appears that many membrane proteins belong to superfamilies sharing a common ancestral gene. Three of these superfamilies include the glycine, GABA, and nicotinic acetylcholine receptors, which are ligand-gated ion channels; the voltage-gated Ca 2÷, K + , and Na ÷ ion channels; and the fl-adrenergic and muscarinic acetylcholine receptors. 61 This last family also includes rhodopsin. Identification of the conserved regions which define membership in these superfamilies has provided scientists with a strategy for identifying and isolating new membrane proteins. First, an oligonucleotide probe is synthesized which matches a conserved sequence. This probe is then used in hybridization studies to identify closely related genes. After obtaining the sequence to the gene, a peptide is synthesized (10-20 amino acids) based on this sequence, and then used to prepare polyclonal antiserum. The antibodies from this serum can be used to determine the subcellular localization of the protein and to purify the protein by immunoaffinity ~methods. As an alternative method, the cloned gene can be attached in frame to another gene, such as fl-galactosidase or protein A, and this fusion gene can be expressed in E. coll. Either of the fusion proteins produced by this method can be easily isolated and used to prepare antisera. In this way, previously unidentified proteins can be isolated based on their homology to known proteins. In a variation of this method, Zinn et al. identified a 6o K.-S. Huang, H. Bayley, M.-J. Liao, E. London, and H. G. Khorana, J. Biol. Chem. 256, 3802 (1981). 61 C. F. Stevens, Nature (London) 328, 198 (1987).

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membrane protein from grasshopper (fasciclin 1), which is important for neurite outgrowth. 6z They wished to find a similar protein in Drosophila so that the function of fasciclin could be studied by genetic deficiency analysis. When antibodies against grasshopper fasciclin 1 did not crossreact with Drosophila proteins, this group successfully used an oligonucleotide probe to identify the gene for the putative Drosophila protein. It is now common practice to identify and isolate genes based on their linkage to genetic diseases. Orkin identified a gene believed to be linked to chronic granulomatosis. 63 Analysis of the sequence identified several hydrophobic regions and putative glycosylation sites, indicating that the gene product was an integral membrane glycoprotein. Antibodies were prepared against a synthetic peptide and then used to identify the protein as cytochrome b from the NADPH oxidase system. Methods of Sample Concentration and Analysis Methods of protein concentration, analysis, and quantitation play an important role in every purification protocol. These are general methods which apply to both soluble and membrane proteins. However, the application of these methods to detergent-solubilized membrane proteins can pose numerous problems and these are discussed below.

1. Methods of concentrating samples: At present there are no concentration methods which are widely applied to integral membrane proteins. In most cases, concentration methods are avoided. Proteins can be solubilized at relatively high concentrations (5-10 mg protein/mi), and methods such as gel filtration or gradient centrifugation, which require small concentrated fractions, may then be used as the initial purification step. Subsequent steps use methods such as affinity chromatography and ion exchange, which can be used with dilute samples. These latter methods also have the effect of concentrating samples. If concentration methods must be used then efforts should be made to avoid dramatic changes in detergent concentrations or protein-detergent ratios. Changes in these conditions can lead to either aggregation or inactivation. Methods of concentration fall into four categories: (a) precipitation, (b) filtration, (c) evaporation, and (d) absorption. a. Precipitation by ammonium sulfate or polyethylene glycol (PEG) is frequently used, but its primary application has been for fractionation rather than concentration. Ice-cold acetone (9 vol/voi sample) or trichloroacetic acid (10-20% w/v) may also be used to precipitate proteins. This harsher method tends to denature proteins. In 62 K. Zinn, L. McAllister, and C. S. Goodman, Cell 53, 577 (1988). 63 S. H. Orkin, T1G 3, 149 (1987).

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addition, precipitation methods do not work well with protein concentrations below 0.1-1 mg/ml. b. Samples may also be concentrated by passing aqueous solutes through a filter membrane which retains the proteins of interest. Solute movement is accelerated by pressure (e.g., Amicon stirred cell), vacuum (e.g., Schleicher and Schuell collodion bag apparatus), or centrifugation (e.g., Amicon Centricon). The collodion bag apparatus and the Micro-ProDiCon (Biomolecular Dynamics) are both capable of simultaneously dialyzing and concentrating samples. Detergents with large micelle sizes (e.g., Triton X-100) will be retained by filters or membranes resulting in the concentration of both protein and detergent. A final problem with filtration is that many membrane proteins form irreversible aggregates on the filter. c. Evaporation and lyophilization are rarely used, although they may be applicable to membrane proteins solubilized in organic solvents. d. Finally, two methods of absorption are occasionally used. In the first case, the sample is placed in a dialysis bag which is then placed in a dry bed of NaCI, sucrose, Sephadex, Ficoll, or PEG. As an alternative, dry Sephadex is added directly to the sample. After the resin has swelled, the concentrated sample is recovered by centrifugation. 2. SDS-PAGE: A widely used method for analysis of chromatographic fractions is polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). 64 In addition, methods for obtaining partial amino acid sequences are now sensitive enough to be performed on proteins eluted from electrophoretic gels. This electrophoresis method may require modification when used with some membrane proteins. SDS may not be sufficient to ensure denaturation, in which case 8 M urea should be included in the sample buffer or in the gel buffers. 65 High concentrations of 2-mercaptoethanol (1-5%) may cause proteolysis. 66 This problem is alleviated by using dithiothreitol (1-2 mM). Standard protocols suggest boiling samples for 3 min prior to electrophoresis. However, some membrane proteins, such as the nicotinic ACh receptor, the muscarinic receptor, the/3-adrenergic receptor, rhodopsin, H+-ATPase, and lactose permease are aggregated by boiling. An alternative is to incubate samples at 25-60 ° for 20-90 min. It may be necessary to increase the concentrations of SDS and reducing agent in the sample buffer. Not all proteases will be inactivated by this treatment, and protease inhibitors may need to be 64 U. K. Laemmli, Nature (London) 227, 680 (1970). 63 C. H. Siu, R. A. Lerner, and W. F. Loomis, J. Mol. Biol. 116, 469 (1977). 66 j. B. C. Findlay, in "Biological Membranes: A Practical Approach" (J. B. C. Findlay and W. H. Evans, eds.), p. 179. IRL Press, Oxford, 1987.

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added to the sample buffer. It is also possible to electrophorese proteins under nondenaturing conditions by using the Laemmli discontinuous buffer system, but replacing SDS with other detergents such as Triton X-100. 67 3. P r o t e i n A s s a y s : One of the most widely used methods for the quantitation of protein is the Lowry assay. 68 However, many of the compounds used in purification of membrane proteins interfere with this assay. 69 Nonionic detergents such as Triton X-100 will precipitate in the Lowry protein assay. These detergents can either be removed by centrifugation or maintained in solution by addition of SDS to the assay. 69 Nonionic detergents do not precipitate in the Bradford and B C A protein assays, which have been proposed as alternatives to the Lowry assay. 7°,7j However, these assays are also susceptible to interference. For example, the BCA assay is susceptible to interference by some lipids. 72 Whichever method is chosen, the influence of detergent- and lipid-containing buffers should be tested on both the assay blank and protein standards (see [6] in this volume for a complete discussion of protein assays). Summary As stated at the beginning of this chapter, it is not possible to present a single step-by-step protocol for the purification of all membrane proteins. We have discussed many of the individual techniques employed to purify membrane proteins and the problems associated with their application. A successful purification protocol will require the use of a combination of these techniques. It should be clear from the examples given, however, that affinity chromatography is by far the most useful technique available. The greatest single obstacle to performing a successful purification is the ability to maintain solubilized proteins in fully dispersed monomeric micelles without inactivating the protein. The best combination of techniques and the optimal conditions for their use can be determined only by trial and error. This process should become easier as a greater number of examples become available and as greater use is made of HPLC and FPLC techniques. 67 C. Bordier, W. F. Loomis, J. Elder, and R. Lerner, J. Biol. Chem. 253, 5133 (1978). O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 255

(1951). 69G. L. Peterson, Anal. Biochem. 100, 201 (1979). 7oM. M. Bradford,Anal. Biochem. 72, 248 (1976). 7~p. K. Smith,R. I. Krohn,G. T. Hermanson,A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. L. Fujimoto,N. M. Goeke, B. J. Goeke, B. J. Olson, and D. C. Kienk,Anal. Biochem. 150, 76 (1985). 72R. J. Kesslerand D. D. Fanestil,Anal. Biochem. 159, 138 (1986).