Isolation and characterization of recombinant human casein kinase II subunits α and β from bacteria

cDNA encoding the casein kinase II (CKII) subunits α and β of human origin were expressed in Escherichia coli using expression vector pT7–7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most of the expressed proteins were produced in an insoluble form. The recombinant CKII α subunit was purified by DEAE‐cellulose chromatography, followed by phosphocellulose and heparin‐agarose chromatography. The recombinant CKII β subunit was extracted from the insoluble pellet and purified in a single step on phosphocellulose. From 10 g bacterial cells, the yield of soluble protein was 12 mg α subunit and 5 mg β subunit. SDS/PAGE analysis of the purified recombinant proteins indicated molecular masses of 42 kDa and 26 kDa for the α and β subunits, respectively, in agreement with the molecular masses determined for the subunits of the native enzyme. The recombinant α subunit exhibited protein kinase activity which was greatest in the absence of monovalent ions. With increasing amounts of salt, α subunit kinase activity declined rapidly. Addition of the β subunit led to maximum stimulation at a 1:1 ratio of both subunits. Using a synthetic peptide (RRRDDDSDDD) as a substrate, the maximum protein kinase stimulation observed was fourfold under the conditions used. The K m of the reconstituted enzyme for the synthetic peptide (80 μM) was comparable to the mammalian enzyme (40–60 μM), whereas the α subunit alone had a K m of 240 μM. After sucrose density gradient analysis, the reconstituted holoenzyme sedimented at the same position as the mammalian CKII holoenzyme.