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

With the advancement of biochemical tools, studies on membrane proteins have grown substantially. Since membrane proteins are difficult to extract and purify, it requires optimization and an orchestrated execution. As described in our basics of membrane protein isolation blog, surface active agents are crucial for the manipulation of membrane proteins. The amphiphillic properties of these components promote the interaction of the membrane proteins, which are wrapped around by hydrophobic lipid bilayer in their native state, to become water soluble. Nonetheless, the solubility cannot be extrapolated into stability and restoration of the native functional structure; therefore, it is not necessary that a detergent (surfactant or a surface-active agent) can yield a suitable stable membrane protein fraction despite good extraction. Also, a detergent that has shown good results previously with a particular membrane protein might not work well with other membrane proteins. In the absence of gold standards or thumb rules for membrane protein extraction, it becomes imperative to understand the physiochemical characteristics of different detergents before extracting the proteins for example, the charge and degree of hydrophobicity of a specific detergent can allow a prediction of its behavior in a solution and interaction with the protein of choice.

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.

The stability of proteins is crucial in many in vitro protein studies and is considered a major requirement in functional studies involving native and recombinant proteins. Thus, understanding protein stability and preserving the native conformation and normal functions of your protein of interest can be very helpful when working with your protein of interest.

Expression of recombinant proteins with peptide or protein tags is widely used in protein research for three main reasons, ease of purification from a large pool of host proteins, enhancing solubility of the protein and for localization studies. Some important steps to be considered while choosing an expression vector are compatibility of tag sequence with that of the desired protein, codon usage, including linker sequences, peptide cleavage sites and the impact of the tag on the nature of desired protein. Various tags are used ranging from large proteins (Maltose Binding Protein {MBP}) to small peptides (Hexa (6X) Histidine). Tags can be added to either N-terminal side or C-terminal side of the desired protein.