Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase gene from Trypanosoma brucei

The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme of Trypanosoma brucei and related parasites provides a rational target for the treatment of African sleeping sickness and several other parasitic diseases. To characterize the T. brucei HGPRT enzyme in detail, the T. brucei hgprt was...

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Veröffentlicht in:Nucleic acids research 1993-11, Vol.21 (23), p.5431-5438
Hauptverfasser: Allen, Thomas E., Ullman, Buddy
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description The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme of Trypanosoma brucei and related parasites provides a rational target for the treatment of African sleeping sickness and several other parasitic diseases. To characterize the T. brucei HGPRT enzyme in detail, the T. brucei hgprt was isolated within a 4.2 kb SalI-KpnI genomic insert and sequenced. Nucleotide sequence analysis revealed an open reading frame of 630 bp that encoded a protein of 210 amino acids with a Mr = 23.4 kd. After gap alignment, the T. brucei HGPRT exhibited 21 - 23 amino acid sequence identity, mostly in three clustered regions, with the HGPRTs from human, S. mansoni, and P. falciparum, indicating that the trypanosome enzyme was the most divergent of the group. Surprisingly, the T. brucei HGPRT was more homologous to the hypoxanthine phosphoribosyltransferase (HPRT) from the prokaryote V. harveyi than to the eukaryotic HGPRTs. Northern blot analysis revealed two trypanosome transcripts of 1.4 and 1.9 kb, each expressed to equivalent degrees in insect vector and mammalian forms of the parasite. The T. brucei hgprt was inserted into an expression plasmid and transformed into Sφ606 E. coli that are deficient in both HPRT and xanthine-guanine phosphoribosyltransferase activities. Soluble, enzymatically active recombinant T. brucei HGPRT was expressed to high levels and purified to homogeneity by GTP-agarose affinity chromatography. The purified recombinant enzyme recognized hypoxanthine, guanine, and allopurinol, but not xanthine or adenine, as substrates and was inhibited by a variety of nucleotide effectors. The availability of a molecular clone encoding the T. brucei hgprt and large quantities of homogeneous recombinant HGPRT enzyme provides an experimentally manipulate molecular and biochemical system for the rational design of novel therapeutic agents for the treatment of African sleeping sickness and other diseases of parasitic origin.
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The T. brucei hgprt was inserted into an expression plasmid and transformed into Sφ606 E. coli that are deficient in both HPRT and xanthine-guanine phosphoribosyltransferase activities. Soluble, enzymatically active recombinant T. brucei HGPRT was expressed to high levels and purified to homogeneity by GTP-agarose affinity chromatography. The purified recombinant enzyme recognized hypoxanthine, guanine, and allopurinol, but not xanthine or adenine, as substrates and was inhibited by a variety of nucleotide effectors. 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To characterize the T. brucei HGPRT enzyme in detail, the T. brucei hgprt was isolated within a 4.2 kb SalI-KpnI genomic insert and sequenced. Nucleotide sequence analysis revealed an open reading frame of 630 bp that encoded a protein of 210 amino acids with a Mr = 23.4 kd. After gap alignment, the T. brucei HGPRT exhibited 21 - 23 amino acid sequence identity, mostly in three clustered regions, with the HGPRTs from human, S. mansoni, and P. falciparum, indicating that the trypanosome enzyme was the most divergent of the group. Surprisingly, the T. brucei HGPRT was more homologous to the hypoxanthine phosphoribosyltransferase (HPRT) from the prokaryote V. harveyi than to the eukaryotic HGPRTs. Northern blot analysis revealed two trypanosome transcripts of 1.4 and 1.9 kb, each expressed to equivalent degrees in insect vector and mammalian forms of the parasite. 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The availability of a molecular clone encoding the T. brucei hgprt and large quantities of homogeneous recombinant HGPRT enzyme provides an experimentally manipulate molecular and biochemical system for the rational design of novel therapeutic agents for the treatment of African sleeping sickness and other diseases of parasitic origin.</abstract><cop>Oxford</cop><pub>Oxford University Press</pub><pmid>8265360</pmid><doi>10.1093/nar/21.23.5431</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record>
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subjects Amino Acid Sequence
Analytical, structural and metabolic biochemistry
Animals
Base Sequence
Biological and medical sciences
Cloning, Molecular
DNA Primers
Enzymes and enzyme inhibitors
Escherichia coli
Fundamental and applied biological sciences. Psychology
Gene Expression
Genes, Protozoan
Hypoxanthine Phosphoribosyltransferase - genetics
Molecular Sequence Data
Protozoan Proteins - genetics
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
RNA, Messenger - genetics
Schistosoma mansoni
Sequence Alignment
Sequence Homology, Amino Acid
Substrate Specificity
Transferases
Trypanosoma brucei
Trypanosoma brucei brucei - genetics
title Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase gene from Trypanosoma brucei
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