Expression, purification, stability optimization and characterization of human Aurora B kinase domain from E. coli |
| |
Authors: | Payal R Sheth Lata Ramanathan Ashwin Ranchod Dianah Barrett Kimberly Gray Rumin Zhang |
| |
Institution: | a Protein Science Department, Merck Research Laboratories, 320 Bent St., Cambridge, MA 02141, USA b Tumor Biology Department, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA c Chemistry Department, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 0Z033, USA |
| |
Abstract: | 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. |
| |
Keywords: | Aurora kinase Cancer Protein purification Protein aggregation Circular dichroism Thermal-shift assay |
本文献已被 ScienceDirect 等数据库收录! |
|