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 (CK2 alpha) and two regulatory subunits (CK2 beta). In higher animals two paralog catalytic chains-abbreviated CK2 alpha and CK2 alpha'-exist which can combine with CK2 beta to three isoforms of the holoenzyme: CK2 alpha beta(2), CK2 alpha'(2)beta(2), and CK2 alpha alpha'beta(2). While CK2 alpha and the "normal" holoenzyme CK2 alpha(2)beta(2) have been extensively characterized in vitro and in vivo, little is known about the enzymological properties of CK2 alpha' and the "alternative" holoenzyme CK2 alpha'(2)beta(2) and about their specific physiological roles. A major reason for this lack of knowledge is the fact that so far CK2 alpha' rather than CK2 alpha has caused serious stability and solubility problems during standard heterologous expression procedures. To overcome them, we developed a preparation scheme for CK2 alpha'(2)beta(2) from Homo sapiens in catalytically active form based on two critical steps: first expression of human CK2 alpha' as a well soluble fusion protein with the maltose binding protein (MBP) and second proteolytic cleavage of CK2 alpha'-MBP in the presence of human CK2 beta so that CK2 alpha' 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 CK2 alpha-based and the CK2 alpha-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. (c) 2005 Elsevier Inc. All rights reserved.