Production and purification of the PEAK pseudokinases for structural and functional studies

The PEAK family of pseudokinases, which comprises PEAK1, PEAK2 and PEAK3, are newly identified scaffolds that dynamically assemble oncogenic signaling pathways known to contribute to the development of several aggressive cancers. A striking feature of this unique family of pseudokinase scaffolds is their large multi-domain structure, which allows them to achieve protein complex assemblies through their structural plasticity and functional versatility. Recent structural advances have begun to reveal the critical regulatory elements that control their function. Specifically, the dimer-dependent scaffolding activity of PEAK pseudokinases is emerging as a critical mechanism for their signaling function, in addition to their ability to hetero-associate to form higher-order regulatory networks to diversify and amplify their signaling output. Here, we present a suite of techniques that enable the efficient expression and purification of PEAK proteins for functional characterization.