Molecular characterization and overexpression of the hypoxanthine-guanine phosphoribosyltransferase gene from Trypanosoma cruzi.
The hypoxanthine-guanine phosphoribosyltransferase (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 structure-based drug design strategy for the treatment of Chagas disease.
Allen
T E
TE
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97201-3098.
Ullman
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D016054:DNA, Protozoan / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014349:Trypanosoma cruzi / Q000201:enzymology*
D017931:DNA Primers; D016054:DNA, Protozoan; D011994:Recombinant Proteins; D007041:Hypoxanthine Phosphoribosyltransferase