Membrane Protein Production in Detergent
Integral membrane proteins are the gateway to the cells. All cells and organelles are encased in an impermeable lipid bilayer and the integral membrane proteins are embedded within the lipid bilayer. Integral membrane proteins are held in the membrane by hydrophobic interactions between the hydrocarbon chains of the lipids and the hydrophobic domains of the proteins. These proteins are the gateways for the entry and exit of many ions, nutrients, waste products, hormones, drugs and large molecules such as proteins and DNA.
In humans ~30% of genes code for integral membrane proteins. They have enormous medical importance. Mutations in membrane proteins are involved in heart diseases, cancers, neurological disorders and cystic fibrosis just to name a few. Unfortunately, integral membrane proteins are difficult to handle and study because they are embedded in the lipid bilayer. Detergents can be used to aid in the careful removal of such membrane proteins.
What are Detergents?
Detergents are critical tools for the study of membrane proteins. They are vital for the isolation and purification of the proteins and are used in the primary solubilization step of reconstitution. They are invaluable in membrane protein recrystallization.
Detergents are soluble amphiphilic molecules consisting of a polar head group and hydrophobic chain (or tail) and exhibit unique properties in aqueous solutions in which they spontaneously form spherical micellar structures. Membrane proteins are frequently soluble in micelles formed by amphiphilic detergents. Detergents solubilize membrane proteins by creating a mock lipid bilayer environment normally inhabited by the protein.
Figure 1. Structure of detergent and formation of micelles
Major Detergent Classifications
According to the different hydrophilic groups of detergent, detergent can be divided into ionic (cationic or anion), non-ionic and zphoteric ionic types.
- Ionic Detergents: Ionic detergents have a polar head that can be either anionic or cationic and a hydrophobic chain or tail with a steroidal backbone. They are very efficient at solubilizing proteins, but almost always cause denaturation of the protein to some extent. An example of an ionic detergent is Sodium Dodecyl Sulfate (SDS).
- Non-Ionic Detergents: Non-ionic detergents have an uncharged hydrophilic head of either Polyoxyethylene or glycosidic group. It is a relatively mild detergent that solubilizes proteins by breaking the lipid-lipid interactions or lipid-protein interactions. Ionic detergents do not break the protein-protein interactions thereby, the solubilized protein is structurally intact in its biological form. Ionic detergents are effective in isolating active membrane proteins. Examples of Non-Ionic detergents are Digitonin and OG.
- Zwitterionic Detergents: The polar head groups of zwitterionic detergents have a neutral charge. They have both ionic and non-ionic properties. The strength of action of zwitterionic detergents is intermediate between both ionic and non-ionic detergents.
Process of Preparing Membrane Protein by Using Detergent
Membrane preparation
Separation of plasma membrane from cells or tissues is the first step in purifying membrane proteins. To prepare cell membrane components, a tissue or cell needs to be homogenized, and the most common method is to homogenize tissue or cell in isotonic sucrose buffer using a Douncehomogenizer. Membrane proteins are relatively stable when integrated in the cell membrane. Protease may be released when cells are broken, so the inactivation caused by proteolytic hydrolysis is the most important problem to be considered during membrane purification. Protease inhibitors are readily available commercially in tablet form. Therefore, 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 that allow them to be separated from other organelles. Differential centrifugation can remove soluble proteins, most of the mitochondria, and the nucleus from the cell homogenate. Sucrose density gradient can further separate cell membranes with different densities.
The solubilization of natural membrane proteins
The membrane protein is embedded in the human lipid double layer. Integrated membrane proteins have at least one protein sequence embedded in the cell membrane. Before purification, the integrated membrane proteins need to solubilize out of the lipid bilayer to become separate proteins. Amphiphilic detergents are commonly used to solubilize integrated membrane proteins from cell membranes. After centrifugation of the membrane treated with detergent, if the membrane protein is in the supernatant part, the membrane protein is considered to be "dissolved" from the membrane. The process by which a stain remover dissolves membrane proteins can be divided into several stages. In the first stage, the detergent binds to the cell membrane. With the increase of detergent content, detergent began to crack the cell membrane. A further increase in the detergent content leads to the formation of a lipid/protein/detergent complex. At this point, the membrane proteins are "dissolved." Additional detergents are required at this time to divide the above complex "degreasing Y d e U p k k t e) into protein/detergents complex and lipid/detergents complex. In general, a ratio of 1 to 2 detergent to protein is sufficient to dissolve the membrane protein into a lipid/albumin/detergent complex, and a ratio of 10 left to right or higher leads to defat of the complex.
Protein purification
Once a suitable decontamination agent has been used to dissolve the membrane albumin from the membrane, the albumin can be labeled by the separation eye. Traditional chromatographic techniques, such as gel filtration, affinity, ion exchange and chromatofocusing, can be used to purify membrane egg white. However, it is necessary to pay attention to several points before the decontamination agent is stored under the strip for chromatographic chromatography.
- Use a sufficient amount of detergent to maintain the integrated membrane proteins in their soluble form in the buffer and prevent protein aggregation.
- Since most detergents are hydrophobic, protein separation methods based on protein hydrophobicity, such as phenyl-agarose gels and reversed-phase chromatography, may not be suitable for membrane protein purification.
- Ionic detergents of solubilizing membrane proteins, such as cholate or deoxycholate, are not suitable for ion exchange chromatography. Non-ionic or facultative detergents can be used in charge-based preparation techniques, including ion exchange chromatography and preparation electrophoresis.
Overall, affinity chromatography is currently the most useful and successful method for purifying integrated membrane proteins and can be used at all purification stages. Because ion-exchange chromatography is sensitive to the ionic strength of the buffer, and gel filtration requires a relatively small volume of concentrated samples, affinity chromatography can be used for purification, concentration, and salt displacement in different chromatographic steps.
Figure 3. Detergent solubilization process of membrane proteins
Beta Lifescience establishes protocols and pipelines for membrane protein expression and purification. With biophysical and biochemical characterization, we offer structural insight into disease targets for both industry and academia.
Detergent Platform Technology Challenges
On the basis of the experience of expression and purification of membrane proteins expressed by detergent technology platform, the selection and combined use of a variety of detergent can effectively increase the content of target transmembrane proteins in total proteins, and ensure the correct and stable conformation. The challenges of the platform are as follows:
- Optimization of detergent concentration: too high detergent concentration may lead to protein denaturation, too low can not effectively dissolve membrane lipids. Therefore, in the process of preparing transmembrane protein, it is necessary to optimize the type and concentration of detergent through experiments.
- Maintenance of protein stability: The detergent sometimes affects the natural conformation and function of the protein, and it is necessary to maintain the appropriate detergent concentration during the purification process, and try to remove or replace the detergent during the final purification step to obtain a functional transmembrane protein.
- Removal of detergent: purified transmembrane proteins often need to be removed for functional studies or structural analysis.
FAQs
Detergents can be denaturing or non-denaturing with respect to protein structure. Denaturing detergents can be anionic such as sodium dodecyl sulfate (SDS) or cationic such as ethyl trimethyl ammonium bromide. These detergents totally disrupt membranes and denature proteins by breaking protein-protein interactions.
The detergent's job is to assist in denaturing the protein and by doing so increase its solubility. The hydrophobic tail of a detergent stabilizes any hydrophobic residues present in the protein and the hydrophilic head disrupts any non-covalent bonds between residues to unfold the protein.
Around 1-2
A detergent/protein ratio of around 1-2 (w/w) is believed to be sufficient to solubilize IMPs to form lipid-protein-detergent mixed micelles. A further increase of detergent concentration causes progressive delipidation of the lipid-protein-detergent mixed micelles.
Ionic detergents can not only disintegrate the membrane and separate it from the hydrophobic part of the membrane protein, but also break the covalent bonds within the protein, or even change the conformation of the hydrophilic part, denaturing the protein, and thus non-ionic detergents are commonly used to obtain biologically active proteins.
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