Engineering and optimization of expression systems for intrinsically disordered proteins

Abstract

Intrinsically disordered proteins (IDPs) are a class of protein or a protein region which is biologically active even in a collapsed or extended state and with a dynamically mobile conformational ensemble. In eukaryotes, about 25-30% of proteins are mostly disordered. Currently, IDP-related is one of the top researches field in the modern protein science due to its recent discovered functions, such as (1) be able to provide flexible linkers between structures proteins and (2) rubber-like entropic springs; (3) containing the sites for post-translational modifications, (4) regulatory protease digestion and (5) for binding to partners such as DNA, tRNA, rRNA, mRNA, proteins or metal ions by changing shape to associate with different targets; (6) containing autoinhibitory domains and (7) signals (as for nuclear localization); and (8) enabling movement through narrow pores. However, one of the most relevant function of IDPs is correlated with therapeutics due to the fact that it can be a very promising target for therapeutic drugs since targeting small molecules to the IDP should enable the development of more effective drug discovery techniques. Although the therapeutic potential of IDPs be completely clear, heterologous expression of IDPs is a field under researched and an optimal expression system for such proteins have not been developed yet: these long proteins show extreme structural heterogeneity and have a complex post-translational modification patterns that are difficult to interpret, beyond the fact that the expression and purification of these proteins are often compromised by their proteolytic sensitivity. In addition, IDPs usually possess a lower rate of synthesis and shorter protein half-lives in comparison to ordered proteins. Professor Pamela Silver group is planning to engineer intrinsically disorder proteins for therapeutic purposes, however, an optimal expression system for such proteins to overcome the challenges of IDPs expression have not yet been developed yet. This project aims to developed an optimal heterologous expression system able to express at high yield and high quality different IDPs for therapeutics. To do so, four different expression system will be tested in order to developed an efficient expression system for IDPs: expression in Pichia pastoris, expression in mammalian cells, expression in a split intein system and expression in a cell-free system. (AU)