Purification, crystallization and drug screening of the IRAK pseudokinases

Pseudokinases are emerging as critical components of cell signaling pathways. Consequently, the ability to obtain large quantities of pure protein for structural characterization and drug discovery efforts has become essential for the study of these proteins. Small molecules binding to pseudokinases may induce allosteric changes and serve as valuable tools to study the physiological roles of these "dead" enzymes. The IRAK family of kinases are key components of the innate immune response and the active IRAK family members, IRAK-1 and -4, have been extensively studied. However, the other two IRAKs, IRAK-2 and IRAK-3, are classified as pseudokinases and their detailed functions and roles remain to be described. In this chapter, we present comprehensive protocols for the purification of IRAKs, the crystallization of the pseudokinase domain of IRAK3, and a high-throughput drug screening pipeline using thermal shift and biolayer-interferometry assays to identify small molecule binders.