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

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

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

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 (US)
Pages (from-to)1231-1238
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume157
Issue number3
DOIs
StatePublished - Dec 30 1988
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Characterization of mutant TMK368K pig citrate synthase expressed in and isolated from escherichia coli'. Together they form a unique fingerprint.

Cite this