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

► Human Aurora B kinase domain buffer optimization using thermal shift assay yielded stable and well-behaved protein amenable for biophysical characterization. ► The binding dissociation constants estimation from thermal shift assays and affinity IC50s from Lanthascreen™ binding assays correlated for inhibitors' binding to the purified Aurora B kinase domain. ► The purified Aurora B kinase domain bound known inhibitors with expected affinity and specificity. ► Aurora B kinase domain bound AZD1152, a selective Aurora B inhibitor, with 10-fold lower affinity than the full-length Aurora B protein although at >10-fold higher affinity than full-length Aurora A protein.