ELLINGTON, W.R.*; BUSH, J.: Cloning and Expression of an Echiuroid Lombricine Kinase

Phosphagen kinases constitute a family of phosphoryl transfer enzymes which are widely distributed throughout the animal kingdom. Among these, creatine kinase (CK) and arginine kinase (AK) have been the most extensively studied. Lombricine kinase (LK) catalyzes the phosphorylation of lombricine (guanidinoethylphospho-O-serine) to lombricine phosphate and back. We report here the cloning of the cDNA and expression of L-lombricine kinase from the innkeeper worm Urechis caupo (an echiuroid). The cDNA was obtained by RTPCR protocols using a universal phosphagen kinase 3’RACE amplification primer. The cDNA consists of 1,830 bp with an open reading frame of 1,101 bp coding for a 366 residue protein. The estimated molecular mass was 40.94 kD, a value consistent with other phosphagen kinase subunits. A full length cDNA was generated by PCR amplification of the original RT product which was ligated into the pET Blue (Novagen) vector followed by transformation of NovaBlue cells and expression of the plasmid in a standard expression host. No soluble LK was expressed but massive amounts of inclusion bodies were produced. Inclusion bodies were harvested, washed and then protein unfolded, refolded and subjected to chromatographic purification. A small amount of soluble, active LK was obtained as validated using a spectrophotometric assay. Analysis of the deduced amino acid sequence of Urechis LK shows that this enzyme displays a number of the key residues thought to be critical in catalysis and provides insights into structural correlates of guanidine specificity. For instance, Urechis LK contains deletions in a flexible loop region which accommodate the large molecular size of the guanidine substrate in the catalytic pocket. (Supported by NSF grant IBN-9631907 to WRE).