Expression, purification, characterization and crystallization of non- and phosphorylated states of JAK2 and JAK3 kinase domain

Journal Article · Tue May 29 00:00:00 EDT 2012 · Protein Expres. Purif.

DOI:https://doi.org/10.1016/j.pep.2009.09.011· OSTI ID:1037899

Hall, Troii; Emmons, Thomas L; Chrencik, Jill E; Gormley, Jennifer A; Weinberg, Robin A; Leone, Joseph W; Hirsch, Jeffrey L; Saabye, Matthew J; Schindler, John F; Day, Jacqueline E; Williams, Jennifer M; Kiefer, James R; Lightle, Sandra A; Harris, Melissa S; Guru, Siradanahalli; Fischer, H David; Tomasselli, Alfredo G ^[1]

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1. Pfizer

Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as viable therapeutic targets in inflammation and oncology related diseases. To date, targeting JAK proteins with highly selective inhibitor compounds have remained elusive. We have expressed the active kinase domains for both JAK2 and JAK3 and devised purification protocols to resolve the non-, mono- (Y1007) and diphosphorylated (Y1007 and Y1008) states of JAK2 and non- and monophosphorylated states of JAK3 (Y980). An optimal purified protein yield of 20, 29 and 69 mg per 20 L cell culture was obtained for the three JAK2 forms, respectively, and 12.2 and 2.3 mg per 10 L fermentation for the two JAK3 forms allowing detailed biochemical and biophysical studies. To monitor the purification process we developed a novel HPLC activity assay where a sequential order of phosphorylation was observed whereby the first tyrosine residue was completely phosphorylated prior to phosphorylation of the tandem tyrosine residue. A Caliper-based microfluidics assay was used to determine the kinetic parameters (K{sub m} and k{sub cat}) for each phosphorylated state, showing that monophosphorylated (Y1007) JAK2 enzyme activity increased 9-fold over that of the nonphosphorylated species, and increased an additional 6-fold for the diphosphorylated (Y1007/Y1008) species, while phosphorylation of JAK3 resulted in a negligible increase in activity. Moreover, crystal structures have been generated for each isolated state of JAK2 and JAK3 with resolutions better than 2.4 {angstrom}. The generation of these reagents has enabled kinetic and structural characterization to inform the design of potent and selective inhibitors of the JAK family.

• Research Organization: Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
• Sponsoring Organization: INDUSTRY
• OSTI ID: 1037899
• Journal Information: Protein Expres. Purif., Vol. 69, Issue (1) ; 01, 2010; ISSN 1046-5928
• Country of Publication: United States
• Language: ENGLISH

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
60 APPLIED LIFE SCIENCES
CRYSTAL STRUCTURE
CRYSTALLIZATION
DESIGN
DISEASES
ENZYME ACTIVITY
FERMENTATION
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
INFLAMMATION
KINETICS
L CELLS
LYMPHOKINES
MONITORS
PHOSPHORYLATION
PHOSPHOTRANSFERASES
PROTEINS
PURIFICATION
RESIDUES
TARGETS
TYROSINE