Purification and characterization of the CK2α′-based holoenzyme, an isozyme of CK2α: A comparative analysis

Protein kinase CK2 (former name: “casein kinase 2”) is a pivotal and ubiquitously expressed member of the eukaryotic protein kinase superfamily. It predominantly exists as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) and two regulatory subunits (CK2β). In higher animals two paralog catalytic chains—abbreviated CK2α and CK2α′—exist which can combine with CK2β to three isoforms of the holoenzyme: CK2α2β2, CK2α2′β2, and CK2αα′β2CK2αα′β2. While CK2α and the “normal” holoenzyme CK2α2β2 have been extensively characterized in vitro and in vivo, little is known about the enzymological properties of CK2α′ and the “alternative” holoenzyme CK2α2′β2 and about their specific physiological roles. A major reason for this lack of knowledge is the fact that so far CK2α′ rather than CK2α has caused serious stability and solubility problems during standard heterologous expression procedures. To overcome them, we developed a preparation scheme for CK2α2′β2 from Homo sapiens in catalytically active form based on two critical steps: first expression of human CK2α′ as a well soluble fusion protein with the maltose binding protein (MBP) and second proteolytic cleavage of CK2α′–MBP in the presence of human CK2β so that CK2α′ subunits are incorporated into holoenzyme complexes directly after their release from MBP. This successful strategy which may be adopted in comparably difficult cases of protein/protein complex preparation is presented here together with evidence that the CK2α′-based and the CK2α-based holoenzymes are similar concerning their catalytic activities but are significantly different with respect to some well-known CK2 properties like autophosphorylation and supra-molecular aggregation.