Trypanosoma brucei ornithine decarboxylase: Enzyme purification, characterization, and expression in Escherichia coli

M. A. Phillips, P. Coffino, C. C. Wang

Research output: Contribution to journalArticlepeer-review

40 Scopus citations


Ornithine decarboxylase from the American trypanosome is an important target for antitrypanosomal chemotherapy. Despite this, the enzyme had not been previously purified or extensively characterized as it is a very low level protein. In this paper we describe the purification of Trypanosoma brucei ornithine decarboxylase from bloodstream form trypomastigotes by 107,000-fold to a specific activity of 2.7 x 106 nmol CO2/h/mg of protein in the parasite. T. brucei ornithine decarboxylase had a native molecular weight of 90,000 and a subunit molecular weight of 45,000. The isoelectric point of the protein was 5.0. The K(m) for ornithine was 280 μM and the K(i) for the irreversible inhibitor α-difluoromethylornithine (DFMO) was 220 μM with a half-time of inactivation at saturating DFMO concentration of 2.7 min. T. brucei ornithine decarboxylase appears similar to mouse ornithine decarboxylase, further supporting our previous suggestion that the selective toxicity of DFMO to the parasite is not due to catalytic differences between the two proteins. Although a small quantity of T. brucei ornithine decarboxylase was purified from T. brucei, extensive structural and kinetic studies will require a more ample source of the enzyme. We therefore expressed our previously cloned T. brucei ornithine decarboxylase gene in Escherichia coli using a vector that contains an inducible λ promoter. T. brucei ornithine decarboxylase activity was induced in E. coli to levels that were 50 to 200 fold of that present in the long-slender bloodstream form of T. brucei. Ornithine decarboxylase activity in the crude E. coli lysate was 1500-6000 nmol of CO2/h/mg of protein and represented 0.05-0.2% of the total cell protein. The recombinant T. brucei ornithine decarboxylase was purified to apparent homogeneity from the transformed E. coli. The purified recombinant enzyme had kinetic and physical properties essentially identical to those of the native enzyme.

Original languageEnglish (US)
Pages (from-to)17933-17941
Number of pages9
JournalJournal of Biological Chemistry
Issue number34
StatePublished - 1988

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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