The B isozyme of creatine kinase is active as a fusion protein in Escherichia coli: in vivo detection by 31P NMR.
A cDNA encoding the B isozyme of creatine kinase (CKB) has been expressed in Escherichia coli from a fusion with lacZ carried by lambda gt11. Western blots indicate that a stable polypeptide with the appropriate mobility for the beta-galactosidase-creatine kinase (beta-gal-CKB) fusion protein cross-reacts with both beta-gal and CKB antiserum. No significant CK activity is detected in control E. coli; however, extracts from cells containing the lambda gt11-CKB construct have a CK activity of 1.54 +/- 0.07 mumol/min per mg protein. The fusion protein appears to provide this activity because immunoprecipitation of protein with beta-gal antiserum leads to a loss of CK activity from extracts. That the enzyme is active in vivo was demonstrated by detection of a phosphocreatine (PCr) peak in the 31P NMR spectrum from E. coli grown on medium supplemented with creatine. As in mammalian brain and muscle, the PCr peak detected was sensitive to the energy status of the E. coli.
Koretsky
A P
AP
Department of Biological Science, Carnegie Mellon University, Pittsburgh, PA 15213.
Traxler
B A
BA
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D003001:Cloning, Molecular; D003402:Creatine Kinase / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007527:Isoenzymes; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D051379:Mice; D010758:Phosphorus; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010758:Phosphorus; D003402:Creatine Kinase