Production of intrinsically disordered proteins for biophysical studies: Tips and tricks

Abstract

Intrinsically disordered proteins (IDPs) have no single, fixed tertiary structure, yet they take on many vital functions in biology. In recent years, considerable effort has been put into the structural characterization of their conformational ensembles, to understand the link between the transient, short- and long-range organizations of IDPs and their functions. Such biophysical studies require substantial amounts of pure protein, representing a major bottleneck in the studies of IDPs. However, the unique physicochemical properties resulting from their compositional bias may be exploited for simple yet effective purification strategies. In this chapter, we provide tips and tricks for IDP production and describe the most important analyses to carry out before bringing an IDP of interest to the laboratory. We outline four purification protocols utilizing the unique properties of IDPs as well as some commonly encountered challenges and pitfalls.