Expression And Purification Of A Stable, Monomeric Creatine Kinase

Abstract

Three isoform gene families of creatine kinase (CK) are present in animals. Two of these, mitochondrial and cytoplasmic CKs, are obligate oligomers . There is substantial evidence for functional interaction between subunits. Attempts at generating active, monomeric CKs have failed or in one case produced ephemerally active but unstable monomers. A third CK isoform, the so-called flagellar CK, is monomeric but is composed of three complete, contiguous CK domains. Each of these domains is catalytically competent but there is clear interaction between active sites (Hoffman et al., FEBS J 275: 646-654 [2008]). In the present effort, we have used a flagellar CK expression construct as a platform to engineer, express and purify a single domain, monomeric CK. Boundaries between the three domains (D1, D2 and D3) were identified by comparison of key catalytic residues and predicted secondary structural elements. A cDNA coding for D2 was amplified by PCR and inserted into an expression vector. Subsequent expression and purification yielded a recombinant CK which was stable as evidenced by the retention of activity over several weeks. Size exclusion chromatography showed that this CK was monomeric as expected, with a mass similar to the predicted Mr based on the amino acid composition. The engineering of a monomeric CK in the present effort clearly shows that oligomerization is not required for catalysis. Conventional wisdom supports the view that CKs evolved from a related phosphotransferase, arginine kinase (AK). AKs are typically monomeric. It seems likely that oligomerization occurred later in the evolution of CKs perhaps due to the selective pressure for targeting to and binding in intracellular compartments. (Supported by NSF grant IOB-0542236 to WRE).