Molecular characterization and overexpression of the hypoxanthine-guanine phosphoribosyltransferase gene from Trypanosoma cruzi

Thomas E. Allen, Buddy Ullman

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

The hypoxanthine-guanine phosphoribosyltransferase gene (HGPRT) enzyme in Trypanosoma cruzi is a rational target for the treatment of Chagas disease. To evaluate the T. cruzi HGPRT in detail, the HGPRT gene (hgprt) was cloned from a genomic library of T. cruzi DNA and sequenced. Translation of the nucleotide sequence of the hgprt revealed an open reading frame of 663 bp that encoded a 25.5-kDa polypeptide of 221 amino acids. The T. cruzi HGPRT exhibited only 24%, 25%, and 21% amino acid sequence identity to its human, Plasmodium falciparum and Schistosoma mansoni counterparts, respectively, but was 50% identical to the T. brucei HGPRT protein. Northern analysis of T. cruzi RNA revealed a 1.8-kb hgprt transcript, while Southern blots of genomic DNA suggested that hgprt was a single copy gene within the T. cruzi genome. The T. cruzi hgprt was inserted into the pBAce expression plasmid and transformed into Escherichia coli that are deficient in hypoxanthine and guanine phosphoribosylating activities. High levels of soluble, enzymatically active T. cruzi HGPRT were obtained, and this expression complemented the bacterial phosphoribosyltransferase deficiencies. The recombinant HGPRT was purified to apparent homogeneity by GTP-agarose affinity chromatography and recognized hypoxanthine, guanine, and allopurinol, but not adenine or xanthine, as substrates. The availability of the hgprt clone and large amounts of pure HGPRT protein provide a foundation for a structurebased drug design strategy for the treatment of Chagas disease.

Original languageEnglish (US)
Pages (from-to)233-245
Number of pages13
JournalMolecular and Biochemical Parasitology
Volume65
Issue number2
DOIs
StatePublished - 1994

Fingerprint

Hypoxanthine Phosphoribosyltransferase
Trypanosoma cruzi
Genes
Hypoxanthine
Chagas Disease
Guanine
Agarose Chromatography
Allopurinol
Xanthine
Genomic Library
Schistosoma mansoni
Drug Design
Adenine
Plasmodium falciparum
Southern Blotting
Affinity Chromatography
Open Reading Frames
Amino Acid Sequence
Proteins
Plasmids

Keywords

  • Drug design
  • Hypoxanthine-guanine phosphoribosyltransferase
  • Molecular cloning
  • Phosphoribosyltransferase
  • Purine salvage
  • Trypanosoma cruzi

ASJC Scopus subject areas

  • Molecular Biology
  • Parasitology

Cite this

Molecular characterization and overexpression of the hypoxanthine-guanine phosphoribosyltransferase gene from Trypanosoma cruzi. / Allen, Thomas E.; Ullman, Buddy.

In: Molecular and Biochemical Parasitology, Vol. 65, No. 2, 1994, p. 233-245.

Research output: Contribution to journalArticle

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