Polyionic Tags as Enhancers of Protein Solubility in Recombinant Protein Expression

Abstract

Since the introduction of recombinant protein expression in the second half of the 1970s, the growth of the biopharmaceutical field has been rapid and protein therapeutics has come to the foreground. Biophysical and structural characterisation of recombinant proteins is the essential prerequisite for their successful development and commercialisation as therapeutics. Despite the challenges, including low protein solubility and inclusion body formation, prokaryotic host systems and particularly Escherichia coli, remain the system of choice for the initial attempt of production of previously unexpressed proteins. Several different approaches have been adopted, including optimisation of growth conditions, expression in the periplasmic space of the bacterial host or co-expression of molecular chaperones, to assist correct protein folding. A very commonly employed approach is also the use of protein fusion tags that enhance protein solubility. Here, a range of experimentally tested peptide tags, which present specific advantages compared to protein fusion tags and the concluding remarks of these experiments are reviewed. Finally, a concept to design solubility-enhancing peptide tags based on a protein's pI is suggested.

Keywords: peptide tag; polyanionic; polycationic; protein fusion tag; protein solubility; recombinant protein expression.

Figure 1

Suggested mechanism of native protein folding and association of intermediate, partially folded protein molecules, bearing native-like secondary structures, leading to inclusion body formation.

Figure 2

A method to enhance protein solubility during recombinant protein production is the introduction of solubility-enhancing tags (protein or peptide) in the recombinant plasmid. By having a few potential mechanisms of action, protein tags can cover a wider range of proteins in order to enhance solubility and most of them act simultaneously as solubility and purification tags. However, peptide tags are more versatile and smaller in size, which means their removal is not always essential, they do not pose a burden on the host system’s metabolism and they do not affect the target protein’s structure or function.