Expression of Torpedo californica creatine kinase in Escherichia coli and purification from inclusion bodies

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 a...

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Bibliographic Details
Published in:Protein expression and purification 2002-10, Vol.26 (1), p.89-95
Main Authors: Wang, Pan-Fen, Novak, Walter R.P, Cantwell, John S, Babbitt, Patricia C, McLeish, Michael J, Kenyon, George L
Format: Article
Language:English
Subjects:
Animals
Circular Dichroism
Creatine Kinase - chemistry
Creatine Kinase - genetics
Creatine Kinase - isolation & purification
Creatine Kinase - metabolism
Electrophoresis, Polyacrylamide Gel
Escherichia coli - genetics
Escherichia coli - metabolism
Gene Expression
Inclusion Bodies - chemistry
Isoelectric Focusing
Kinetics
Mass Spectrometry
Protein Denaturation
Protein Renaturation
Torpedo
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Summary: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 54 mg 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.
ISSN:1046-5928
1096-0279
DOI:10.1016/S1046-5928(02)00512-0