Recombinant expression, purification, and characterization of Toxoplasma gondii adenosine kinase

John A. Darling, William J. Sullivan, Darrick Carter, Buddy Ullman, David S. Roos

Research output: Contribution to journalArticlepeer-review

44 Scopus citations


Toxoplasma gondii lacks the capacity to synthesize purines de novo, and adenosine kinase (AK)-mediated phosphorylation of salvaged adenosine provides the major route of purine acquisition by this parasite. T. gondii AK thus represents a promising target for rational design of antiparasitic compounds. In order to further our understanding of this therapeutically relevant enzyme, an AK cDNA from T. gondii was overexpressed in E. coli using the pBAce expression system, and the recombinant protein was purified to apparent homogeneity using conventional protein purification techniques. Kinetic analysis of TgAK revealed K(m) values of 1.9 μM for adenosine and 54.4 μM for ATP, with a k(cat) of 26.1 min-1. Other naturally occurring purine nucleosides, nucleobases, and ribose did not significantly inhibit adenosine phosphorylation, but inhibition was observed using certain purine nucleoside analogs. Adenine arabinoside (AraA), 4-nitrobenzylthioinosine (NBMPR), and 7-deazaadenosine (tubercidin) were all shown to be substrates of T. gondii AK. Transgenic AK knock-out parasites were resistant to these compounds in cell culture assays, consistent with their proposed action as subversive substrates in vivo. Copyright (C) 1999 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)15-23
Number of pages9
JournalMolecular and Biochemical Parasitology
Issue number1
StatePublished - Sep 20 1999
Externally publishedYes


  • Apicomplexan parasites
  • Enzyme kinetics
  • Nucleoside metabolism
  • Protein purification
  • Purine salvage

ASJC Scopus subject areas

  • Parasitology
  • Molecular Biology


Dive into the research topics of 'Recombinant expression, purification, and characterization of Toxoplasma gondii adenosine kinase'. Together they form a unique fingerprint.

Cite this