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 AurB69-333 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 AurB69-333. 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 AurB69-333 fragment is a good surrogate for its full-length counterpart for biophysical and structural analyses. © 2010 Elsevier Inc.
CITATION STYLE
Sheth, P. R., Ramanathan, L., Ranchod, A., Basso, A. D., Barrett, D., Zhao, J., … Le, H. V. (2010). Expression, purification, stability optimization and characterization of human Aurora B kinase domain from E. coli. Archives of Biochemistry and Biophysics, 503(2), 191–201. https://doi.org/10.1016/j.abb.2010.08.004
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