Optimizing the Production of Aurora Kinase A and Validation of Constructs with different Sequential Lengths

Sammanfattning: Aurora Kinase A is a kinase involved in multiple signaling pathways and interactions during mitosis, making it an essential kinase that deregulated causes cancer diseases in affected patients. Structural research shows mainly static snapshots of possible conformations of the partly disordered protein. This is due to challenges in generating a monodisperse pure sample with high stability enough for dynamic biophysical measurements. Optimizing the production of Aurora A and validating constructs with different sequential lengths using light scattering techniques, thermal stability screening, mass characterization, mass spectrometry, and immunoassay techniques is important for future structural insights useful for drug discoveries. In this project, validation of constructs concluded that no significant difference in cleavage of His-tag, purification possibilities, monodispersity nor stability is shown by variate start residue from 118-122 to end residue 403. Expression of an Aurora Kinase A constructs with sequential length 118-403 is preferred to be executed at 18 degrees, otherwise, temperature differences during expression show no impact on produced Aurora A. Magnesium chloride has been shown to have an impact on stability where a higher concentration stabilizes Aurora Kinase A. Moreover, concentration differences of NaCl were shown to not affect the stability of Aurora A. During this project a polydisperse sample was generated and has given insights into Aurora A´s behavior in solution.