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The B isozyme of creatine kinase is active as a fusion protein in Escherichia coli : In vivo detection by 31 P NMR
Author(s) -
Koretsky Alan P.,
Traxler Beth A.
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)81206-2
Subject(s) - creatine kinase , escherichia coli , fusion protein , microbiology and biotechnology , isozyme , phosphocreatine , antiserum , complementary dna , biochemistry , biology , in vivo , enzyme , chemistry , antibody , gene , energy metabolism , recombinant dna , endocrinology , immunology
A cDNA encoding the B isozyme of creatine kinase (CK B ) has been expressed in Escherichia coli from a fusion with lacZ carried by λgt11. Western blots indicate that a stable polypeptide with the appropriate mobility for the β‐galactosidase‐creatine kinase (β‐gal‐CK B ) fusion protein cross‐reacts with both β‐gal and CK B antiserum. No significant CK activity is detected in control E. coli ; however, extracts from cells containing the λgt11‐CK B construct have a CK activity of 1.54±0.07 μmol/min per mg protein. The fusion protein appears to provide this activity because immunoprecipitation of protein with β‐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 31 P 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 .