Abstract
JAK kinases are critical mediators in development, differentiation, and homeostasis and accordingly, have become well-validated targets for drug discovery efforts. In recent years, the integration of X-ray crystallography in kinase-focused drug discovery programs has provided a powerful rationale for chemical modification by allowing a unique glimpse of a bound inhibitor to its target. Such structural information has not only led to an improved understanding of the key drivers of potency and specificity of several JAK-specific compounds but has greatly facilitated and accelerated the design of compounds with improved pharmacokinetic properties.
JAK kinases are traditionally difficult candidates to express in significant quantities, generally requiring eukaryotic expression systems, protein engineering, mutations to yield soluble, homogeneous samples suitable for crystallization studies. Here we review the key methods utilized to express, purify, and crystallize the JAK kinases and provide a detail description of the methods that we have developed to express, purify, and crystallize recombinant JAK1 and JAK2 proteins in the presence of small molecule inhibitors.
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Acknowledgments
This work was supported by the Australian Research Council and the National Health and Medical Research Council Industry Fellowship (I.L.). We thank Dr Onisha Patel and Dr Neal Williams for conducting part of the experiments described in this chapter. We thank Prof Jamie Rossjohn and Prof Andrew Wilks for supporting the research.
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Lucet, I.S., Bamert, R. (2013). Production and Crystallization of Recombinant JAK Proteins. In: Nicholson, S., Nicola, N. (eds) JAK-STAT Signalling. Methods in Molecular Biology, vol 967. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-242-1_20
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DOI: https://doi.org/10.1007/978-1-62703-242-1_20
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