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

While several protein separation technologies have been developed in recent years, two-dimensional gel electrophoresis remains one of the most popular techniques used in the field of proteomics. This is not surprising since 2D PAGE is routinely used to accurately analyze the individual components of even the most complex proteins, and can simultaneously resolve more than 5000 proteins (depending on the gel size used).

Recombinant production of proteins involves transfecting cells with desired gene in a DNA vector. The gene then translates into a protein using host cellular machinery. These expressed proteins can then be extracted by lysing the cells ans subsequent purification steps. Both Prokaryotic and Eukaryotic expression systems are widely used . Each system has its own advantages and disadvantages. A particular expression system is chosen depending on economic and qualitative aspects, such as the type of protein, function and desired protein yield.

The cloning and expression of proteins to product recombinant proteins is a hugely popular technique that allows for the production of copious quantities of protein that almost mimic native proteins. Although a popular and useful technique, one of the main reasons for its use can also be a drawback.  In some cases, over-expression of the recombinant protein leads to an accumulation of misfolding protein that aggregates and is sequestered into inclusion bodies.   The purification of inclusion bodies is time consuming, involves harsh denaturants, detergents and other chemicals, resulting in damaged and denatured proteins.  If a researcher is successful in extracting these proteins then they must invest more time and effort to refold the denatured proteins. For this reason, researcher's want to prevent their recombinant proteins from ending up in inclusion bodies.

Living entities have a tightly regulated dynamic system, where majority of the energy expenditure goes for maintaining the homeostasis. Most of the biological processes such as growth, development, metabolism etc are regulated at the crucial control point of protein translation and degradation (rate of protein turnover). Studies on different organisms/model systems have suggested that transcriptome modifies itself dynamically in response to different cellular conditions. The translational regulation is a slightly independent add-on layer of control after that, hence, during the cell processes a linear relationship of transcriptome and translatome is relatively difficult to establish. The efficacious identification of lesser abundant proteins is of lower magnitude, albeit of having sub-femtomolar level of sensitivity of modern state-of-the- art Mass spectroscopic instruments because of a narrow dynamicity and sequencing speed.