Rapid method for evaluation of CK2 enzymatic activity and CK2α/CK2β-interaction in Escherichia coli cell lysates

Gast, Alexander; Schreiber, Sebastian; Jose, Joachim

Zusammenfassung

This study introduces a novel, rapid assay to measure CK2α activity in Escherichia coli cell lysates. By fusing CK2α with the fluorescent protein mScarlet it was possible to quantify CK2α concentration directly in lysates. We used the dose-dependent increase of CK2α activity after addition of CK2β1-193 to determine the dissociation constants (K D ) of the CK2α/CK2β-interaction. As a first trial, activity and affinity of the variant CK2αR191Q to CK2β1-193 was investigated using the developed assays. This mutation in the CSNK2A1 gene, encoding CK2α is related to the Okur-Chung Neurodevelopmental Syndrome (OCNDS). Apparent K D values of 13 nM for the CK2αR191Q/CK2β interaction and 7.4 nM for the CK2α/CK2β interaction were determined using nonlinear regression. Uncertainties with regards to the concentration of both binding partners were propagated through the entire process of nonlinear regression by Monte Carlo simulations. This way, accuracy confidence intervals of the K D -values were derived. This resulted in 96.4 % confidence that the accurate K D -values of the CK2α-CK2β and CK2αR191Q-CK2β interactions were different. The results suggest potential disruptions in oligomeric assembly caused by the R191Q mutation.