HOFFMAN, G.G.*; ELLINGTON, W.R.; Florida State Univ.; Florida State Univ.: Bacterial expression of a functional, octameric mitochondrial creatine kinase from a polychaete.

Creatine kinase (CK), which catalyzes the reversible phosphorylation of creatine, is widely distributed in lophotrochozoan protostomes and deuterostomes. Multiple gene duplication events have led to the production of isoforms which may be expressed tissue-specifically, assume different quaternary structures and/or be targeted to specific intracellular compartments. In primitive-type spermatozoa of many invertebrates, the CK system is compartmentalized consisting of mitochondrial, membrane-associated octamers (MiCK) and a unique contiguous trimeric CK in the flagellum. We have previously shown that the spermatozoa of the polychaete Chaetopterus variopedatus express an MiCK which displays a high degree of similarity to mammalian and avian MiCKs in terms of octameric structure, pI, and amino acid sequence (Ellington et al., FEBS Lett. 425: 75-78; Pineda and Ellington, Eur. J. Biochem. 264: 67-73). Unlike its vertebrate counterparts, Chaetopterus MiCK octamers are highly stable and interact very tightly with mitochondrial membranes. In order to probe via site-directed mutagenesis the role of specific residues in quaternary structure and membrane binding, we have generated the full-length cDNA from total RNA of this polychaete, ligated it into the pETBlue-1 (Novagen) expression vector, and transformed E. coli (DE3) hosts. Expression of this construct yielded minimal soluble protein when performed at 37�C, but significant amounts of active MiCK were obtained when expression took place at 15�C. A two-step purification procedure involving cation exchange and gel filtration yielded an essentially homogeneous preparation that was very stable, octameric and displayed high activity.