Expression, purification, stability optimization and characterization of human Aurora B kinase domain from E. coli

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Aurora B kinase is crucial for cell division (mitotic progression).
• Dysregulation of Aurora B kinase is implicated in cancer (tumorigenesis).
• Structural and binding data for human Aurora B kinase inhibitors are limited.

Purpose of the Study:

• To identify a human Aurora B kinase construct suitable for large-scale production.
• To enable biophysical and structural studies of Aurora B kinase and its inhibitors.
• To optimize conditions for protein stability and minimize aggregation.

Main Methods:

• Expressed and purified a human Aurora B kinase fragment (AurB(69-333)).
• Utilized thermal-shift assay for high-throughput screening of 192 conditions.
• Performed direct ligand binding analyses using temperature-dependent circular dichroism (TdCD) and Lanthascreen™ assay.
• Correlated binding affinities with enzymatic activity (IC50 values).

Main Results:

• The AurB(69-333) construct expressed well but required optimization for stability.
• Thermal-shift assay identified optimal pH and salt conditions to enhance protein stability.
• TdCD and Lanthascreen™ assays confirmed the purified protein was folded and functional.
• Binding affinity rank-order correlated with enzymatic IC50 values for most inhibitors.

Conclusions:

• The optimized AurB(69-333) fragment is stable and functional.
• This fragment serves as a reliable surrogate for full-length Aurora B kinase in biophysical studies.
• The findings facilitate structural and binding analyses of Aurora B kinase inhibitors, aiding cancer research.