Characterization of mutant TMK368K pig citrate synthase expressed in and isolated from escherichia coli

Claudia T. Evans, Daniel D. Owens, Clive A. Slaughter, P. Srere

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

A cDNA that encodes pig citrate synthase (PCS) was inserted into a plasmid T7 vector and was expressed in an E. coli gltA mutant. Up to 10 mg of purified PCS was obtained from 2 liters of E. coli. The mammalian protein produced in E. coli comigrated with the enzyme purified from pig heart on a SDS-polyacrylamide gel (SDS-PAGE) with an Mr of 50,000, and reacted with a polyclonal antibody directed against pig heart citrate synthase. The Vmax and Km of the expressed PCS were indistinguishable from those of the pig heart enzyme. The PCS produced in E. coli did not contain the trimethylation modification of Lys 368, characteristic of the pig heart enzyme. These data suggest that the PCS protein produced in E. coli is catalytically similar to the enzyme purified from pig heart and methylation of Lys 368 is not essential for catalysis.

Original languageEnglish
Pages (from-to)1231-1238
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume157
Issue number3
DOIs
Publication statusPublished - Dec 30 1988

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Citrate (si)-Synthase
Escherichia coli
Swine
Enzymes
Methylation
Catalysis
Proteins
Plasmids
Complementary DNA
Antibodies

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Characterization of mutant TMK368K pig citrate synthase expressed in and isolated from escherichia coli. / Evans, Claudia T.; Owens, Daniel D.; Slaughter, Clive A.; Srere, P.

In: Biochemical and Biophysical Research Communications, Vol. 157, No. 3, 30.12.1988, p. 1231-1238.

Research output: Contribution to journalArticle

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