Robust approach for production of the human oncology target Aurora kinase B in complex with its binding partner INCENP.

Protein kinases are key players in many eukaryotic signal transduction cascades and are as a result often linked to human disease. In humans, the mitotic protein kinase family of Aurora kinases consist of three members: Aurora A, B and C. All three members are involved in cell division with proposed implications in various human cancers. The human Aurora kinase B has in particular proven challenging to study with structural biology approaches, and this is mainly due to difficulties in producing the large quantities of active enzyme required for such studies. Here, we present a novel and E. coli -based production system that allows for production of milligram quantities of well-folded and active human Aurora B in complex with its binding partner INCENP. The complex is produced as a continuous polypeptide chain and the resulting fusion protein is cleaved with TEV protease to generate a stable and native heterodimer of the Aurora B:INCENP complex. The activity, stability and degree of phosphorylation of the protein complex was quantified by using a coupled ATPase assay, 31P NMR spectroscopy and mass spectrometry. The developed production system enables isotope labeling and we here report the first 1H–15N-HSQC of the human Aurora B:INCENP complex. Our developed production strategy paves the way for future structural and functional studies of Aurora B and can as such assist the development of novel anticancer drugs targeting this important mitotic protein kinase. • Design of a cleavable human INCENP-Aurora B fusion protein allows for robust expression in E. coli. • Purification of heterogeneously phosphorylated, stable and active Aurora B:INCENP on a mg-scale. • The Aurora B:INCENP complex can phosphorylate the native full-length human histone H3. • Reports on the first heteronuclear 2D NMR spectrum of the human Aurora B:INCENP complex. • The presented novel production system creates an avenue for future structural studies. [ABSTRACT FROM AUTHOR]