The pET17 expression vector was used to express creatine kinase from the electric organ of Torpedo californica as inclusion bodies in Escherichia coli BL21 (DE3) cells. The insoluble aggregate was dissolved in 8 M urea and, following extraction with Triton X-100 the enzyme was refolded by dialysis against Tris buffer (pH 8.0) containing 0.2 M NaCl. After two buffer changes., chromatography on Blue Sepharose was used as a final step in the purification procedure. Approximately 54mg active protein was recovered from a 1 L culture and the refolded enzyme had a specific activity of 75 U/mg. The molecular mass of the purified protein was consistent with that predicted from the amino acid sequence and the CD spectrum of the refolded enzyme was essentially identical to that of creatine kinase from human muscle (HMCK). The K-m values of ATP and ADP were also similar to those of HMCK, while the K-m values for both phosphocreatine and creatine were approximately 5-10-fold higher. The purification described here is in marked contrast with earlier attempts at purification of this isozyme where, in a process yielding less than 1 mg/L culture, enzyme with a specific activity of ca. 5 U/mg was obtained. (C) 2002 Elsevier Science (USA). All rights reserved.