Expression, purification, characterization and crystallization of non- and phosphorylated states of JAK2 and JAK3 kinase domain.
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 69mg per 20L cell culture was obtained for the three JAK2 forms, respectively, and 12.2 and 2.3mg per 10L 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(m) and k(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 2.4A. The generation of these reagents has enabled kinetic and structural characterization to inform the design of potent and selective inhibitors of the JAK family.
Pfizer Inc., Global Research and Development, St. Louis Laboratories, St. Louis, MO 63017-1732, USA.
Troii
T
Hall
Thomas L
TL
Emmons
Jill E
JE
Chrencik
Jennifer A
JA
Gormley
Robin A
RA
Weinberg
Joseph W
JW
Leone
Jeffrey L
JL
Hirsch
Matthew J
MJ
Saabye
John F
JF
Schindler
Jacqueline E
JE
Day
Jennifer M
JM
Williams
James R
JR
Kiefer
Sandra A
SA
Lightle
Melissa S
MS
Harris
Siradanahalli
S
Guru
H David
HD
Fischer
Alfredo G
AG
Tomasselli
D000595:Amino Acid Sequence; D055162:Biocatalysis; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D006801:Humans; D053614:Janus Kinase 2 / Q000737:chemistry* / Q000302:isolation & purification*; D053616:Janus Kinase 3 / Q000737:chemistry* / Q000302:isolation & purification*; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary
D053614:Janus Kinase 2; D053616:Janus Kinase 3