Biosynthesis and site-specific labeling of CK2 proteins

The biosynthesis and purification of CK2α-pAzF was performed as described before [42 (link)]. For the recombinant expression of CK2β^1-193-pAzF, plasmids pCK2β^{1-193,Y176Stop} and pEVOL-pAzF were used to transform E. coli BL21(DE3). CK2β^1-193-pAzF was obtained and purified referring to the process of CK2α-pAzF [42 (link)] with the exception that before purification the cell lysate was stirred overnight at 4 °C in order to extract CK2β^1-193-pAzF [54 (link)].
Purified CK2α-pAzF (130 µg/mL) in buffer P50 (25 mM Tris/HCl (pH 8.5), 50 mM NaCl) was incubated with 50 µM DBCO-Sulfo-Cy5 (Jena Bioscience, Jena, Germany) for 1 h in the dark at room temperature (RT). By SPAAC reaction the specific labeled CK2α-DBCO-Sulfo-Cy5 was obtained. For the site-specific labeling of CK2β^1-193, purified CK2β^1-193-pAzF in buffer P100 (25 mM Tris/HCl (pH 8.5), 100 mM NaCl) was treated with fluorescein alkyne (0.25 mM), TCEP (Tris(2-carboxyethyl)phosphine, 1 mM), TBTA (Tris(benzyltriazolylmethyl)amine, 0.17 mM) and CuSO₄ (1 mM) for for 1 h in the dark at RT. By CuAAC reaction the specific labeled CK2β^1-193-Flu was obtained. For MST measurements an additional ultrafiltration step using vivaspin500 columns (Sartorius, Göttingen, Germany) was used to remove unbound fluorophore and additives of the click reaction.