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

Abstract

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 phosphorylationmore » 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.« less

Authors:
; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ;  [1]
  1. Pfizer
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
INDUSTRY
OSTI Identifier:
1037899
Resource Type:
Journal Article
Journal Name:
Protein Expres. Purif.
Additional Journal Information:
Journal Volume: 69; Journal Issue: (1) ; 01, 2010; Journal ID: ISSN 1046-5928
Country of Publication:
United States
Language:
ENGLISH
Subject:
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

Citation Formats

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, and Tomasselli, Alfredo G. Expression, purification, characterization and crystallization of non- and phosphorylated states of JAK2 and JAK3 kinase domain. United States: N. p., 2012. Web. doi:10.1016/j.pep.2009.09.011.
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. Expression, purification, characterization and crystallization of non- and phosphorylated states of JAK2 and JAK3 kinase domain. United States. https://doi.org/10.1016/j.pep.2009.09.011
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, and Tomasselli, Alfredo G. 2012. "Expression, purification, characterization and crystallization of non- and phosphorylated states of JAK2 and JAK3 kinase domain". United States. https://doi.org/10.1016/j.pep.2009.09.011.
@article{osti_1037899,
title = {Expression, purification, characterization and crystallization of non- and phosphorylated states of JAK2 and JAK3 kinase domain},
author = {Hall, Troii and Emmons, Thomas L and Chrencik, Jill E and Gormley, Jennifer A and Weinberg, Robin A and Leone, Joseph W and Hirsch, Jeffrey L and Saabye, Matthew J and Schindler, John F and Day, Jacqueline E and Williams, Jennifer M and Kiefer, James R and Lightle, Sandra A and Harris, Melissa S and Guru, Siradanahalli and Fischer, H David and Tomasselli, Alfredo G},
abstractNote = {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.},
doi = {10.1016/j.pep.2009.09.011},
url = {https://www.osti.gov/biblio/1037899}, journal = {Protein Expres. Purif.},
issn = {1046-5928},
number = (1) ; 01, 2010,
volume = 69,
place = {United States},
year = {Tue May 29 00:00:00 EDT 2012},
month = {Tue May 29 00:00:00 EDT 2012}
}