Date Published: February 4, 2016
Publisher: Springer Berlin Heidelberg
Author(s): Ajamaluddin Malik.
Disulfide bonds occurred in majority of secreted protein. Formation of correct disulfide bonds are must for achieving native conformation, solubility and activity. Production of recombinant proteins containing disulfide bond for therapeutic, diagnostic and various other purposes is a challenging task of research. Production of such proteins in the reducing cytosolic compartment of E. coli usually ends up in inclusion bodies formation. Refolding of inclusion bodies can be difficult, time and labor consuming and uneconomical. Translocation of these proteins into the oxidative periplasmic compartment provides correct environment to undergo proper disulfide bonds formation and thus achieving native conformation. However, not all proteins can be efficiently translocated to the periplasm with the help of bacterial signal peptides. Therefore, fusion to a small well-folded and stable periplasmic protein is more promising for periplasmic production of disulfide bonded proteins. In the past decades, several full-length proteins or domains were used for enhancing translocation and solubility. Here, protein fusion tags that significantly increase the yields of target proteins in the periplasmic space are reviewed.
Since the advent of production of recombinant proteins, application of therapeutic and diagnostic proteins as biopharmaceuticals was changed remarkably (Walsh 2014). These proteins are required in huge amount and usually can not be obtained from natural sources due to extremely low availability. Moreover, Genetically engineered proteins with special benefits (e.g. Insulin analogs) are as such molecules which can therefore only be obtained via recombinant technology (Walsh 2000, 2006; Sanchez and Demain 2012). Escherichia coli was the first and still popularly used host for the fast and economical production of recombinant proteins (Vincentelli and Romier 2013; Chance et al. 1981; Choi and Lee 2004; Rosano and Ceccarelli 2014; Lebendiker and Danieli 2014). In-depth knowledge of genetic and biochemical pathways of E. coli and availability of variety of vectors made is an attractive host for such purposes. Although significant improvements have been made at transcription, translation and translocation, still obtaining soluble and bioactive proteins is a major challenge (Pines and Inouye 1999; Baneyx 1999; Rosano and Ceccarelli 2014).
Every protein is unique and due to their different applications such as academic research, diagnostic or therapeutic usage, the quantity and purity level vary. Therefore, no single fusion tag will address every requirement. Fusion tags are helpful in enhancing their solubility and stability. Protein fusion tag with μM-nM ligand affinity generally results in 90–99 % purity after affinity chromatography. Removal of protein fusion tag and producing recombinant protein with authentic N terminal adds another layer of complexity. When considering which protein fusion to use, important queries should keep in mind such as: nature of protein itself, how much protein required, application of protein, is fusion tag removal necessary or not, how much additional residues could be tolerated at N terminal? To remove most part of the fusion protein, highly specific protease cleavage site (TEV protease, thrombin, enterokinase, etc.) could be placed in the linker region between fusion tag and model protein. Also, non-specific proteases such as trypsin could be used to generate authentic N terminus as demonstrated in the case of Ecotin-proinsulin fusion protein (Malik et al. 2007). If authentic N terminus is must for the application, ubiquitin fusion technology could be used as successfully demonstrated in ecotin–ubiquitin–peptide fusion system (Paal et al. 2009).