Purification experiments of membrane proteins
Purification experiments of membrane proteins
Membrane proteins play a key role in biological processes. They are involved in cell-cell and cell-matrix interactions, cell organelle and cell formation, transport of ions, metabolites and proteins across the plasma membrane, and transport of RNA across the nuclear membrane. Given the importance of membrane proteins in a variety of cellular functions, about 50 % of the drugs currently available for various diseases target them.
Author: Burgess et al., Translated by Wei Chen, this experiment is from "Protein Purification Guide".
Operation method
Purification experiments of membrane proteins Move Isolation of plasma membranes from cells or tissues is the first step in purifying membrane proteins. Due to the lack of biochemical methods that can effectively isolate membrane proteins solubilized by detergents, investing some time in purification of the plasma membrane components will be beneficial to the results of the subsequent steps. Most membrane proteins are low in content, so it is important to select tissues or cell lines that are readily available in large quantities and can express the target membrane proteins at high levels. Recently, there has been a growing interest in the use of cell surface proteins as markers for the identification of different cell lines or stem cells. As one of the implications, how to obtain sufficient amount of cell membranes from a limited number of cells and how to enrich the enzymatic activity of plasma membrane markers has become a new concern in the field of membrane separation. The preparation of cell membrane components requires the breaking up of tissues or cells, and the most common method is to homogenize the tissues or cells in isotonic sucrose buffer (0.25 m d/L, p H 7~8) using a Douncehomogenizer. Membrane proteins are relatively stable when integrated within the cell membrane. Whereas proteases may be released when cells are broken, inactivation due to protein hydrolysis is the most important consideration in the membrane purification process. Cocktail mixes of protease inhibitors are commercially available in convenient tablet form, such as complete protease inhibitor cocktail mix [complete protease inhibitor Cocktail (R o c h e ,Indianapolis, I N )]. The extracellular structural domains of membrane proteins are in direct contact with the oxidizing environment , and most of the sulfhydryl groups are present in the form of disulfide bonds, which are formed when proteins are processed in the endoplasmic reticulum. Therefore, reducing agents may not be necessary in this step. In fact, high concentrations of reducing agents can alter the conformation of existing disulfide bonds, resulting in inactivation of membrane protease activity or loss of ligand binding activity. The extracellular domains of membrane proteins are relatively resistant to protein hydrolysis due to the disulfide bond structure and glycosylation modifications. However, there are exceptions. For example, the Ca~-dependent cell adhesion molecule, Cadhermus, is susceptible to protein hydrolysis when Ca2+ is removed by EDTA. In contrast, the membrane-mediated The most commonly used membrane separation method uses a combination of differential centrifugation and sucrose density gradient centrifugation. Due to differences in lipid and protein composition, cell membranes have different densities allowing them to be separated from other organelles. Differential centrifugation removes soluble proteins, most of the mitochondria, and the nucleus from the cell homogenate. Sucrose density gradients can further separate cell membranes of different densities. However, multistep centrifugation is too lengthy and only a small fraction of the plasma membrane can be obtained. Generally much of the plasma membrane is lost in the earlier centrifugation steps. Therefore, this method is more suitable for isolating cell membranes from more readily available tissues. In biochemical studies, rat liver is one of the most commonly used tissues for isolation of plasma membranes. A number of methods have been developed to isolate different membrane components from rat liver. The method used by Neville ( 1%8), which involves homogenizing the liver in a hypotonic solution followed by discontinuous sucrose gradient centrifugation, is very good at recovering liver plasma membranes and has been widely used. In cases where only a small number of tissue culture cells are used, there is a need to increase the recovery of plasma membrane proteins without sacrificing membrane purity. Affinity matrices provide a simple and rapid method for membrane purification. Traditional agarose or acrylamide affinity matrices cannot be used to isolate cell membranes because they precipitate relatively dense organelles (e.g., nuclei). Chemically treated magnetic beads can be coupled to a variety of proteins, which has become a new form of affinity matrix. Unlike traditional agarose or acrylamide affinity matrices, magnetic beads can be easily separated from the mixture with a magnet and can therefore be used to separate organelles independent of their density. By simply placing the centrifuge tube containing the magnetic beads close to the magnet, the beads are recovered at the end of the centrifuge tube close to the magnet and are easily separated from the mixture. Thus, magnetic beads can be used as an alternative to centrifugation. This property can be advantageous for separating plasma membranes from other organelles. We have recently purified membrane proteins from cultured epithelial cells using magnetic beads with immobilized lectins (Leeetal. ,2008). This procedure takes advantage of the fact that some membrane proteins are glycosylated and can bind lectin-glycoprotein In this procedure, the biotinylated lectin associated with concanavalin A (ConA) binds to streptavidin magnetic beads, thereby immobilizing ConA on the beads. The ConA-containing beads are mixed with homogenized cell lysate, and the beads are recovered at the end of the centrifuge tube to remove other organelles that do not bind to the beads. The 5' nuclease, a membrane protein, showed enhanced activity after recovery of the magnetic beads with ConA compared to total cell lysates from prostate PC-3 cells or cervical HeLa cells, thus indicating that the cell membrane was bound to the ConA magnetic beads. A drawback of the lectin magnetic bead method is that we were unable to elute the cell membrane from the ConA magnetic beads using the competing sugar a-methylmannose (alpha-methyl mannoside). This may be because the competitive sugar cannot enter the binding site between the cell membrane and the ConA magnetic beads. Therefore, we used a decontaminating agent to solubilize the membrane proteins from the ConA magnetic beads. Solubilization using decontaminants Membrane proteins are embedded in lipid bilayers. Integral membrane proteins have at least one protein sequence embedded in the cell membrane, whereas peripheral proteins are associated with the cell membrane through electrostatic or, in some cases, hydrophobic interactions. A high salt or high p H solution division is used to dissociate peripheral membrane proteins from the membrane (Schindler et aL ,2006), e.g. 0-5m o l / L N a C l . Since no detergent is used in this process, peripheral membrane proteins can be purified in a similar way to soluble proteins. Prior to purification, integral membrane proteins need to be solubilized from the lipid bilayer, thus becoming separate proteins. Amphipathic detergents are commonly used to solubilize integral membrane proteins from cell membranes. There are perhaps three types of detergents: ionic, nonionic, and zwitterionic. Chapter 41 of this book discusses the different types of detergents. Therefore, we will only discuss the use of detergents in membrane protein purification. After centrifugation of detergent-treated cell membranes at 105,000 g for I h at 4°C, membrane proteins are considered to be "solubilized" from the cell membrane if they are in the supernatant fraction. This process of membrane protein solubilization by the detergent can be divided into several stages. In the first stage, the detergent binds to the cell membrane. As the amount of detergent increases, the detergent begins to cleave the cell membrane. Further increase in the amount of detergent leads to the formation of lipid/protein/detergent complexes. At this point, the membrane proteins are "solubilized". Additional detergent is required to "degrease" the complexes into protein/detergent complexes and lipid/detergent complexes. In general, a detergent-to-protein ratio of 1 to 2 is sufficient to solubilize membrane proteins into lipid/protein/detergent complexes, with ratios of about 10 or more resulting in degreasing of the complexes. The choice of detergent can be simply stated as the selection of a detergent that will work on the target protein. Non-denaturing detergents are capable of solubilizing membrane proteins without inactivation or loss of function. Of the available detergents, Triton X-100, sodium cholate, CHAPS, and octylglucoside are, in most cases, non-denaturing. The presence of detergents can affect protein purification in several ways. Decontaminants affect the detection of protein activity, e.g. they affect the translocation activity of cell membrane transport proteins, and for receptors, they affect their ligand binding activity. Since the proteins are no longer associated with the cell membrane, measurement of transport activity requires reassembly of soluble membrane proteins into phospholipid vesicles. And for specific receptors, methods that can separate unbound ligands from ligand-receptor complexes are also required. These requirements may limit the type of detergent used in solubilization. For example, if there is a subsequent need to recombine membrane proteins into phospholipid vesicles, detergents with high critical micellar concentrations (sodium cholate, CHAPS, octylglucoside) should be used because they are more readily removed by dialysis. Some types of detergents interfere with some of the protein assays. Derivatives of polyoxyethylene, such as Triton X-100, C 12E 9 , and t w e e n series produce false positives in the Thomas Brilliant Blue 0 2 5 0 dye-binding measurements (also known as the Bradford assay) (Bradford, 1976). Sodium cholate or sodium deoxycholate produces a precipitate in the Bradford assay, and the Triton X-100 and NP-40 absorb at 280 nm and thus interfere with the UV absorption method of monitoring eluted proteins in chromatography. This requires a change in the protein detection method or selection of a decontaminant compatible with the protein detection method. The bicinchoninic acid (BCA) protein assay (Smith et al., 1985) is compatible with some detergents. A modified method based on the Lowry protein assay (Peterson, 1977), in which proteins are first precipitated from solution by deoxycholate and trichloroacetic acid (T C A ), avoids the interference of detergents and allows the protein concentration to be measured throughout the purification process. However, the modified Lowry method takes longer than the Bradford method. In some cases, the purity of the detergent is important. For example, impurities in the detergent can interfere with X-ray structural analysis or mass spectrometry. Polyoxyethylene derivatives such as Tnton X-100, LubroI PX, tween, and the Brij series contain polymers of various lengths and are chemically heterogeneous. Octyl glucoside, dodecyl maltoside and CHAPS, on the other hand, have very well-defined chemical compositions and can be obtained with relatively high purity. Nonionic detergents, including polyoxyethylene derivatives such as Triton X-100 and the tween series, are not as effective as ionic detergents in dissociating protein complexes, but may not be as effective as ionic detergents. Screening for detergents capable of solubilizing integral membrane proteins must begin with identifying those detergents that can solubilize but not inactivate membrane proteins. After these requirements are met, other factors need to be considered, including compatibility with protein testing, identification and chromatographic methods. The interference of detergents with various analytical methods is discussed in Section 4 of this chapter. To screen the detergent, a membrane component is prepared in a specific protein concentration, such as I mg/mL, and then a detergent solution is added in varying concentrations from 0.2 to 20 mg/mL. Solubilization usually occurs at a detergent/protein ratio of 0-1 to 10 (w/w). The solution is incubated at ○ ~ 4°C for 30 ~ 60 min, followed by centrifugation at 4°C at 105,000 g for I h. The solution is then incubated with the supernatant and the egg white. The activity of the target protein is then examined in the supernatant and sediment. If most of the activity appears in the supernatant, it means that the detergent is suitable for the membrane protein. If the activity is not present in the supernatant but is present in the starch, then the detergent is not able to dissolve the protein from the cell membrane or not enough detergent has been added. If the activity is not present in both the supernatant and the starch, then the detergent cannot dissolve the protein from the cell membranes or the amount of detergent added is insufficient. Once the membrane proteins are solubilized from the cell membranes with suitable detergents, the target proteins can be separated. Traditional chromatographic techniques such as gel filtration, affinity, ion exchange and chromatofocusing can be used for membrane protein purification. However, the use of chromatography in the presence of detergents requires the following considerations. (1) Use a sufficient amount of detergent to maintain integral membrane proteins in a soluble form in the buffer and to prevent protein aggregation. (2) Protein separation methods based on protein hydrophobicity, such as phenyl-agarose gels and reversed-phase chromatography, may be unsuitable for purification of membrane proteins because of the hydrophobicity of most detergents. (3) Ionic detergents that solubilize membrane proteins, such as cholate or deoxycholate, are not suitable for use in ion exchange chromatography. Nonionic or partially decontaminating agents can be used in charge-based preparation techniques, including ion exchange chromatography and preparative electrophoresis. (4) Decontaminants containing sugar groups may interfere with specific lectin chromatography, e.g., octylglucoside interferes with ConA chromatography. (5) Membrane proteins have a greater apparent molecular mass in gel filtration due to the fact that dissolved membrane proteins are in the detergent micelles. A detergent that forms large molecular mass micelles, such as Trtion X-100, increases the molecular mass of solubilized membrane proteins by 60 to 100 kDa. As a result, most proteins are found in the high molecular mass fraction, which makes separation of proteins based on molecular size difficult. (6) Binding of membrane proteins to detergent micelles, especially detergents with large micelle sizes or that are themselves ionic, can mask the charge of membrane proteins. Thus, ion exchange chromatography may not be as capable of separating membrane proteins as non-membrane proteins. (7) Overall, affinity chromatography is by far the most useful and successful method for purifying integral membrane proteins and can be used at all stages of purification. Because ion-exchange chromatography is sensitive to the ionic strength of the buffer and gel filtration requires a relatively small volume of concentrated sample, affinity chromatography can be used for purification, concentration, and salt replacement in different chromatographic steps . Several commonly used affinity chromatography methods for membrane protein purification are described in the following sections. 4.1 Lectin Affinity Chromatography There are 3 types of affinity chromatography using general ligands (e.g., lectins), specific ligands (e.g., enzyme inhibitors, hormones), and antibodies, respectively. Immobilized lectins are a common form of affinity chromatography. Lectins are sugar-binding proteins that can be used to rapidly and gently purify plasma membrane glycoproteins. Lectin-glycan interactions are reversible and can be inhibited by monosaccharides. Since membrane proteins are often glycosylated, lectin chromatography is useful for purifying membrane proteins. A large number of lectins have been identified , the most widely used of which are ConA [conjugated dextro-mannose ct (crI> mannose)] and wheat germ lectin [germ The following is an example of an experimental protocol for purifying membrane proteins using a lectin column. (1) Transfer 2 mL of wheat germ agglutinin (WGA)-agarose to a disposable plastic column (PolyPrep, Bio-RadLaboratories) and wash the WGA-agarose affinity matrix. The column was filled with 100 mL of wash buffer [50 mmol/LHEPES (p H 7.0, 0.1 % detergent], and the wash solution was allowed to flow through the column to remove unbound WGA and preservation buffer. (2) Add washed WGA-agarose to the dissolved cell membrane extract. Rotate at room temperature or oscillate for 30 min to mix the WGA-agarose with the soluble cell membrane extract. (3) Centrifuge at 600 r/m in for I m in to precipitate the WGA-agarose. Pipette out the supernatant. Add 10 times the volume of wash solution (based on the volume of WGA-agarose) and invert several times. (4) Repeat step (3) twice. (5) Add 2 m L of Wash Solution to W G A - Agarose and transfer W G A - Agarose back to the column. Wash with 5 to 10 times the column volume of Wash Solution. Perform a protein assay at regular intervals, such as with the Thomas Brilliant Blue Protein Assay until the protein level drops to a background value, i.e., similar to the protein level in the wash solution. (6) Add half the column volume of elution buffer, e.g., 50 mmol/L H E PE S (PH 7. 4), 0. 1 % decontaminant, 0-25 m illion I/L N -acetylaminoglucose, to elute the glycoproteins from W G A -agarose. The eluted fraction was collected. (7) Add half of the column volume of elution buffer and collect the eluted fraction. (8) Repeat step (7) 4 times. (9) Detect the protein concentration in the eluate. For example, aspirate 5 pL of each eluate and assay the protein concentration by the Cauloblue method. Pool the appropriate fractions together. 4.2 Ligand Affinity Chromatography The key to successful ligand affinity chromatography is that the affinity between the ligand and the receptor is strong enough to allow for binding and washing during the purification step. Ligands are usually immobilized onto an affinity support by coupling. The affinity of the ligand (or inhibitor) to the receptor may be altered due to the presence of a detergent. Therefore, if a ligand affinity column is used for purification, it is critical to select a detergent that does not significantly reduce the affinity activity of the receptor and ligand. 4.3 Antibody Affinity Chromatography The use of immobilized antibodies to specific membrane proteins is the most powerful method for purifying membrane proteins, if antibodies are available. Antibodies are relatively stable in non-ionic detergents and are therefore compatible with detergents in solubilized cell membrane preparations. The difficulty lies in eluting membrane proteins from immunoaffinity columns (see Chapter 28 for details). If the detergent is completely removed, most membrane proteins will aggregate and precipitate. Therefore, in order to maintain the function of membrane proteins, it is common to remove one detergent while introducing another. In some cases, such as during the initial solubilization phase, excess detergent in the sample can interfere with protein activity and concentration measurements, and it is necessary to remove the excess detergent. In other cases, the initial detergent used for solubilization may not be suitable for subsequent chromatographic or analytical steps, and it may be necessary to replace it with another detergent. For example, ionic detergents are not compatible with ion exchange chromatography and isoelectric focusing and need to be replaced with non-ionic or concurrent detergents. If membrane proteins need to be reassembled into phospholipid vesicles, it is necessary to change to a detergent with a high critical micellar concentration so that the membrane proteins can be reassembled into phospholipid vesicles by removing the detergent through analytical methods. The ease of removal of the desiccant is highly dependent on its nature. Detergents with high critical micelle concentrations (>1 mmol/L), such as bile salts and octylglucoside, are readily removed by dialysis or ultrafiltration. Foulants with low critical micelle concentrations (<1 mmol/L), such as non-ionic fouling agents Triton X-100, C12E9, Brij, Tween, are difficult to remove by analysis, and can be removed by analytical matrix adsorption, gel filtration, and equilibration. See Chapter 41 for methods of removing and replacing detergents. Expression and purification of recombinant integral membrane proteins are often required to obtain adequate amounts of protein for studies of protein structure or for the production of antibodies against membrane proteins. Membrane proteins are usually expressed in mammalian or insect cells. Signal sequences are very important for targeting protein to the endoplasmic reticulum and affect protein synthesis and post-translational modification. Therefore, labels are usually added at the end of the sequence to avoid affecting the membrane targeting process of the protein. In some cases, signaling sequences from other proteins have been Examples of expression and purification of membrane proteins can be found in several papers. One example is the expression and purification of cell adhesion molecules from insect cells (CEACAMKPhanetaL, 2001). In this study, the C-terminal end of the expressed integral membrane protein was labeled with seven histones and the original signal sequence was used, and it was found that the recombinant CEACAM1 protein was localized in the cell membrane. The cells were lysed and the membranes were solubilized with Triton X-100, and the His-labeled CEACM1 was purified in a single step by metal affinity chromatography. For more product details, please visit Aladdin Scientific website.
I. Membrane Preparation
protein signaling is usually susceptible to protein hydrolysis. The receptor tyrosine kinase activity of the insulin receptor, for example, will be dramatically lost if sufficient amounts of protease inhibitors are not present during homogenization and subsequent membrane separation steps.
membrane proteins is described in Section 3 of this chapter.
(Hjelmeland, 1990). For specific membrane proteins, the optimal ratio of detergent to protein for solubilizing membrane proteins needs to be determined experimentally.
In most cases, octylglucoside, sodium cholate, CHAPS, and octylglucoside are non-mutagenic, although some of their activity is lost during solubilization.
Many proteins are more stable in nonionic detergents.
If activity is not detected in either the supernatant or the starch, then the detergent has inactivated the target protein.
agglutinin, binding sialic acid and acetylglucosamine (3(sialic acid and ^ I> GIcNAc)]. Several aspects of affinity chromatography using lectins need to be mentioned here. First , the ability of the target protein to bind to a specific lectin needs to be experimentally verified. Since different tissues have different glycosylases, sometimes the same glycoprotein expressed in different tissues of the same animal may have different lectin binding specificities. Secondly, affinity chromatography of specific lectins purifies a set of glycoproteins with a specific glycosylation type, and therefore does not achieve the specificity of ligand or antibody affinity chromatography.
Secondly, the affinity chromatography of a specific lectin purifies a set of glycoproteins with a specific glycosylation type and therefore does not achieve the degree of purification achieved by ligand or antibody affinity chromatography. Finally, lectins are sensitive to specific types of detergents. While nonionic detergents such as Tritonx-100 (up to 2.5 %, m/V) have a negligible effect on the ligand binding activity of Cognac or wheat germ lectins, some ionic detergents such as SDS may inactivate the lectin.
Sometimes, signaling sequences from other proteins are used to increase the efficiency of membrane targeting. Smaller labels, such as His6 labels or short peptide labels, do not alter protein significantly and can increase the probability of membrane protein expression by heterologous cells. Metallic affinity chromatography is a good choice for expressing and purifying membrane proteins because of its high affinity and freedom from detergents. Other affinity labels such as FLAG, Myc or HA can also be used. However, purification of proteins with solid antibodies is limited and much more costly than metal affinity chromatography.