Expression, purification, stability optimization and characterization of human Aurora B kinase domain from E. coli.
Aurora B kinase plays a critical role in regulating mitotic progression, and its dysregulation has been linked to tumorigenesis. The structure of the kinase domain of human Aurora B and the complementary information of binding thermodynamics of known Aurora inhibitors is lacking. Towards that effort, we sought to identify a human Aurora B construct that would be amenable for large-scale protein production for biophysical and structural studies. Although the designed AurB(69-333) construct expressed at high levels in Escherichia coli, the purified protein was largely unstable and prone to aggregation. We employed thermal-shift assay for high-throughput screening of 192 conditions to identify optimal pH and salt conditions that increased the stability and minimized aggregation of AurB(69-333). Direct ligand binding analyses using temperature-dependent circular dichroism (TdCD) and TR-FRET-based Lanthascreen™ binding assay showed that the purified protein was folded and functional. The affinity rank-order obtained using TdCD and Lanthascreen™ binding assay correlated with enzymatic IC50 values measured using full-length Aurora B protein for all the inhibitors tested except for AZD1152. The direct binding results support the hypothesis that the purified human AurB(69-333) fragment is a good surrogate for its full-length counterpart for biophysical and structural analyses.
Sheth
Payal R
PR
Protein Science Department, Cambridge, MA 02141, USA. payal.sheth@spcorp.com
Ramanathan
Lata
L
Ranchod
Ashwin
A
Basso
Andrea D
AD
Barrett
Dianah
D
Zhao
Jia
J
Gray
Kimberly
K
Liu
Yan-Hui
YH
Zhang
Rumin
R
Le
Hung V
HV
D000595:Amino Acid Sequence; D064107:Aurora Kinase B; D064127:Aurora Kinases; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D020128:Inhibitory Concentration 50; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017510:Protein Folding; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D011993:Recombinant Fusion Proteins; C576921:AURKB protein, human; D064107:Aurora Kinase B; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases