title: Production and crystallization of recombinant JAK proteins.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, VIC, Australia. isabelle.lucet@monash.edu
authors.forename: Isabelle S
authors.initials: IS
authors.lastname: Lucet
authors.forename: Rebecca
authors.initials: R
authors.lastname: Bamert
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D003460:Crystallization / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D053612:Janus Kinases / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D053612:Janus Kinases