Purification Experiment of Membrane Protein

Membrane preparation

Isolation of the plasma membrane from cells or tissues is the first step in purifying membrane proteins. Due to the lack of a biochemical method that effectively separates the membrane protein solubilized by the detergent, investing in the purification of the plasma membrane component for some time would 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 that are highly expressed in the membrane protein of interest. Recently, there has been an increasing interest in the study of cell surface proteins as markers for identifying different cell lines or stem cells. As one of its effects, how to obtain a sufficient amount of cell membrane from a limited number of cells and to enrich the enzymatic activity of the plasma membrane marker has become a new focus in the field of membrane separation.

Preparation of cell membrane components requires disruption of tissue or cells. The most common method is to homogenize tissue or cells in isotonic sucrose buffer (0.25 md / L, p H 7 to 8) using a Douncehomogenizer. dish. Membrane proteins are relatively stable when integrated into the cell membrane. Protease may be released when cells are broken, so the inactivation caused by protein hydrolysis is the most important issue in membrane purification. Commercially available cocktails of protease inhibitors in the form of convenient tablets, such as complete protease inhibitor Cocktail (R o c h e , Indianapolis, I N ). The extracellular domain of membrane proteins is in direct contact with the oxidative environment, and most of the sulfhydryl groups are in the form of disulfide bonds, which are formed when proteins are processed in the endoplasmic reticulum. Therefore, the reducing agent may not be needed in this step. In fact, high concentrations of reducing agents alter the conformation of existing disulfide bonds, resulting in inactivation of membrane protease activity or loss of ligand binding activity. Due to the disulfide bond structure and glycosylation modification, the extracellular domain of membrane proteins is relatively resistant to protein hydrolysis. However, there are exceptions. For example, Ca~-dependent cell adhesion molecule C a d h e r m s is susceptible to protein hydrolysis when C a 2+ is removed by E D T A . In contrast, the intracellular domain that mediates membrane protein signaling is often susceptible to protein hydrolysis. For example, the insulin receptor, if there is not enough protease inhibitor in the homogenate and subsequent membrane separation steps, its receptor tyrosine kinase activity will be greatly lost.

The most common membrane separation method employs a combination of differential centrifugation and sucrose density gradient centrifugation. Due to differences in lipid and protein composition, cell membranes have different densities that allow them to be separated from other organelles. Differential centrifugation removes soluble proteins, most of the mitochondria and nuclei from the cell homogenate. The sucrose density gradient can further separate cell membranes of different densities. However, multi-step centrifugation is too lengthy and only a small fraction of the plasma membrane is obtained. Many plasma membranes are generally lost in earlier centrifugation steps. Therefore, this method is more suitable for separating cell membranes from tissues that are easier to obtain. In biochemical studies, the liver of rats is one of the tissues most commonly used to isolate the plasma membrane of cells. There have been many methods for separating different membrane components from rat liver. Neville (1%8) uses a method of homogenizing the liver in a hypotonic solution followed by a discontinuous sucrose gradient centrifugation, which is a very good method for recovering the liver plasma membrane and has been widely used.

In the case where only a small amount of tissue culture cells are used, it is necessary to increase the recovery of the plasma membrane protein without sacrificing the purity of the membrane. The affinity matrix provides a simple and rapid method of membrane purification. Conventional agarose or acrylamide affinity matrices cannot be used to separate cell membranes because they precipitate relatively high density organelles (such as nuclei). Chemically treated magnetic beads can be coupled to a variety of proteins, which has become a new form of affinity matrix. Unlike conventional agarose or acrylamide affinity matrices, magnetic beads can be easily separated from the mixture by magnets and can therefore be used to separate organelles regardless of the density of the organelles. Simply by placing the centrifuge tube containing the magnetic beads close to the magnet, the magnetic beads can be recovered in the centrifuge tube near the end of the magnet and easily separated from the mixture. Therefore, magnetic beads can be used as an alternative to centrifugation. This property has great advantages for separating the plasma membrane from other organelles. We recently purified membrane proteins from cultured epithelial cells using immobilized magnetic beads (Leeetal., 2008). This step takes advantage of the fact that some membrane proteins are glycosylated and capable of binding lectin-glycoprotein in this process, biotinylated lectin with concanavain A (ConA) and chains The streptavidin magnetic beads are combined to fix ConA to the magnetic beads. The magnetic beads with ConA are mixed with the homogenized cell lysate, and the magnetic beads are recovered at one end of the centrifuge tube to remove other organelles that are not bound to the magnetic beads. The 5' nuclease is a membrane protein whose activity is enhanced after the recovery of ConA magnetic beads compared to the total cell lysate of prostate PC-3 cells or cervical HeLa cells, indicating that the cell membrane binds to ConA magnetic beads. . One drawback of the lectin magnetic bead method is that we are unable to elute cell membranes from ConA magnetic beads using the competitive alpha-methyl mannoside. This may be because competitive sugars cannot enter the binding site between the human cell membrane and the ConA magnetic beads. Therefore, we used a detergent to dissolve the membrane protein from the ConA magnetic beads.

Solubilization of natural membrane proteins

The membrane protein is embedded in the lipid bilayer. The integral membrane protein has at least one protein sequence embedded in the cell membrane, while the peripheral protein is linked to the cell membrane by electrostatic interaction or in some cases by hydrophobic interaction. The high-salt or high-p H solution is used to dissociate the peripheral membrane proteins from the membrane (Schindler et al, 2006), such as 0•5m o / L N a C l . Since no detergent is used in this process, the peripheral membrane proteins can be purified in a similar manner to soluble proteins.

Prior to purification, the integral membrane protein needs to be solubilized from the lipid bilayer to become a separate protein. Amphipathic detergents are commonly used to solubilize integral membrane proteins from cell membranes. Decontaminants may also be classified into three types: ionic, nonionic, and zwitterionic.

After the detergent-treated cell membrane was centrifuged at 105 000 g and 4 ° C for 1 h, if the membrane protein was in the supernatant fraction, the membrane protein was considered to "dissolve" from the cell membrane. This process of detergent membrane proteins can be divided into several stages. In the first stage, the detergent binds to the cell membrane. As the detergent content increases, the detergent begins to lyse the cell membrane. A further increase in detergent content can result in the formation of a lipid/protein/detergent complex. At this time, the membrane protein is "dissolved". At this point, an additional detergent is required to "defatted Y de U pkkte" into a protein/detergent complex and a lipid/detergent complex. In general, the ratio of detergent to protein is 1 to 2 is sufficient to dissolve the membrane protein into a lipid/protein/detergent complex. A ratio of about 10 or higher will result in defatting of the complex.

(Hjelmeland, 1990). For a specific membrane protein, the best decontaminant and protein ratio of the lytic membrane protein needs to be determined experimentally.

The choice of decontaminant can be simply stated as the selection of a detergency that will work for the target protein. Non-deformable detergents can solubilize the membrane without losing it or losing its function. Among the alternative detergents, Triton X-100, sodium cholate, CHAPS, and octylglucoside are non-denatured in most cases, although they are lost during the dissolution process. Part of the activity.

The presence of detergents can affect the purification of proteins in a number of ways. Detergents can affect the detection of protein activity. For example, detergents can affect the transport activity of cell membrane transporters, while for receptors, detergents can affect their ligand binding activity. Since proteins are no longer associated with cell membranes, measuring transport activity requires recombination of soluble membrane proteins into phospholipid vesicles. Also for specific receptors, a method is required to separate the unbound ligand from the ligand-receptor complex. These requirements may limit the type of detergent used in solubilization. For example, if it is necessary to recombine the membrane into a phospholipid vesicle, a detergent with a high critical binder concentration (sodium cholate, CH A P S , octyl glucoside) should be used because they are more easily removed by dialysis.

Purification of membrane proteins

(1) Use a sufficient amount of detergent to maintain the integral membrane protein in a soluble form in the buffer and prevent protein aggregation.

(2) Protein separation methods based on protein hydrophobicity, such as phenyl-sepharose and reversed phase chromatography, may not be suitable for purification of membrane proteins, since most detergents are hydrophobic.

(3) Ionic detergents that solubilize membrane proteins, such as cholate or deoxycholate, are not suitable for ion exchange chromatography. Nonionic or facultative detergents can be used in charge-based preparation techniques, including ion exchange chromatography and preparative electrophoresis.

(4) Glycosyl-containing detergents may interfere with specific lectin chromatography, such as octylglucoside, which interferes with ConA stratification.

(5) Since the dissolved membrane protein is in the detergent micelle, the membrane protein has a larger apparent molecular mass in gel filtration. Detergents capable of forming macromolecular mass micelles, such as Trtion X-100, increase the molecular mass of the dissolved membrane protein by 60 to 100 kDa. Therefore, most proteins appear in the mass portion of the polymer, making protein separation based on molecular size difficult.

(6) The binding of membrane proteins to detergent micelles, especially detergents with large micelle size or ionic species, can mask the charge of membrane proteins. Therefore, the ability of ion exchange chromatography to separate membrane proteins may not be as good as non-membrane proteins.

(7) In general, affinity chromatography is currently the most useful and successful method for purifying integrated membrane proteins and can be used in all stages of purification. Since ion exchange chromatography is sensitive to the ionic strength of the buffer, while gel filtration requires a relatively small volume of concentrated sample, affinity chromatography can be used for purification, concentration, and salt displacement in different chromatography steps. Several commonly used affinity chromatography methods for membrane protein purification are set forth in the following sections.

Protein structure studies or in order to produce antibodies against membrane antibody proteins, it is often necessary to express and purify the recombinant membrane protein to obtain sufficient protein. Membrane is usually expressed as a mammalian cell or a cytoplasmic cell. The signal sequence is important for targeting the protein to the endoplasmic reticulum, which affects the synthesis of the protein and the post-translational modification. Therefore, the label is usually added to the end of the sequence to avoid the film target process that affects the protein. Sometimes, the signal sequence from other proteins is used to increase the efficiency of the custom membrane protein antibody.