The Protein Man's Blog | A Discussion of Protein Research

Proteins are the building blocks of cells and are the most abundant and diverse biomolecules. Proteins are literally responsible for almost all the functional aspect of a cell, from making the cellular membrane to catalyzing a biological reaction. As per the database, approximately 20-30% of the total encoded proteins have integral membrane proteins while 10-20% portion comprises membrane-associated proteins. This diverse group of proteins includes signal transducers, transporters, membrane channels, and cell surface receptors. This specialized group of proteins has become a target for therapeutic drug development in the past few decades. However, due to the unavailability of complete structural details of these proteins, targeting is a little difficult and that makes drug designing trickier than expected. The right approach is to design polished methods that can help the extraction of membrane proteins and their analysis.

Purification of membranous proteins is not as an elementary procedure as that of cytosolic protein extraction. The very first step is extremely delicate and ambiguous which requires disruption of the phospholipid bilayer without harming the protein structure. The most common agent used to meet these expectations is amphiphilic molecules such as detergents that can easily replicate structural substitution of the natural phospholipid molecules. Since the extraction methods have been developed, several classes of detergents were used to optimize the most efficient extraction method. Therefore, detergent screening becomes an essential step for developing membrane protein extraction protocols. However, apart from some general surfactants or detergents, there are no specific guidelines that can suggest which detergent would be the most suitable for a class of membrane proteins. The protocol has to be optimized according to the experimental need.

Strategies for membrane proteins isolation: 

1. Extraction and Solubilization: The source of the proteins (mammalian cells, tissues, bacterial cells etc) are homogenised in a suitable buffer containing protease inhibitors. A detergent containing buffer is used to extract membrane proteins from the lipid bilayer. Different types of detergents, such as ionic detergents, non-ionic detergents, bile salts detergents, and zwitterionic detergents, are available

Factors to be kept in mind for detergent screening:

• It is highly unlikely that a cherry-picked detergent is uniformly superior for all type of membranous proteins.
• Extraction of transmembrane proteins from the lipid layer requires a fine balance and articulation through the properties of the chosen surfactants and the surrounding lipid molecules of the protein. It should be harsh enough to disrupt the bonds between the protein and the surrounding lipids but mild enough to leave the internal stabilizing forces of the protein unperturbed.
• Generally, detergents having a small, charged head groups and comparatively shorter alkyl chains are more stringent and can largely damage the membrane protein, comparatively milder detergents have longer alkyl chain and large, neutral head groups. For e.g., ionic detergents such as SDS, however, is effective but cause significant membrane protein denaturation.  
• The extent of the solubilization and the stability of the membranous depend on the ratio of detergent: protein. Large complexes consisting of membrane lipids and the protein can be achieved at a low ratio (1:10) while with increasing ratio, the complexes become smaller in sizes. When it reaches 10:1 to 20:1, complexes devoid of membrane lipids can be obtained. Nonetheless, this ratio has to be optimized for specific protein type.

 Not just detergent concentration and type, many other factors, including the buffer concentration and temperature of the buffer used for making tissue or cell homogenate, can effect membrane protein extraction. Ionic concentration of the buffer is one of the decisive factors for the solubility of membrane proteins. Hence, nearly 150 mM NaCl is added in the buffers with Polyols to stabilize the solubilized proteins. The most preferable buffer is a phosphate buffer (concentration ranging from 0.1-0.5 M) because of its known protein stabilizing properties. Low concentrations of glycerol should be preferred to avoid viscosity which might hinder removal of salts in ultracentrifugation or on chromatographic columns for getting a purified fraction of membrane proteins.  

2. Removal of detergent:

During the process of extraction to solubilize membrane proteins, an excess of detergents and salts are used, which can create disruption in the further experimental layout. Hence, the detergents and salts have to be cleared to make a purified concoction of the extracted membrane proteins.  Different methods can be employed for purifying the solubilized membrane proteins:

1. Dialysis: This method works best with detergents having a high crtical micelle concetration (cmc) value and a lower ratio of molecular weight/ cross sectional area of the micelle formed. This method cannot be employed if a low cmc value detergent is used such as non-ionic detergent.
2. Hydrophobic adsorption: Hydrophobic resins or beads can behave like a magnet for the detergents because of their amphiphillic properties. This is a suitable method for detergents having a low cmc.
3. Gel Chromatography: The detergent molecules can easily be separated from protein mix based on their sizes using gel chromatography.
4. Ion-exchange chromatography: This method harnesses the amphiphillic nature of the detergent molecules. It can be used to remove ionic and zwitter ionic detergents.
5. Nickel Columns and His tags: This is a useful method to purify membrane proteins that are over expressed with His tags.

 Membrane protein extraction is a challenging task because:

• Membrane proteins are expressed in a very low amount. Therefore, to get a better yield, they need to get over expressed in a suitable host. However, overexpression can be a tedious job as there are possibilities of refolding, truncation or instability.
• Membrane proteins are highly hydrophobic in nature, making them to form aggregates in solution.
• Membrane proteins are difficult in terms of stabilization and maintenance of functional and structural integrity.
• They are more susceptible to protease degradation; therefore require extra attention in stabilization.