TY - JOUR
T1 - Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase gene from Trypanosoma brucei
AU - Allen, Thomas E.
AU - Ullman, Buddy
N1 - Funding Information:
This work was supported by grant AI-23682 from the National Institute of Allergy and Infectious Disease. Thomas Allen was a recipient of an N.L. Tartar Trust Fellowship from the Medical Research Foundation of Oregon. Buddy Ullman is a Burroughs Wellcome Fund Scholar in Molecular Parasitology, and this work was supported in part by a grant from The Burroughs Wellcome Fund.
PY - 1993/11/25
Y1 - 1993/11/25
N2 - 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.
AB - 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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U2 - 10.1093/nar/21.23.5431
DO - 10.1093/nar/21.23.5431
M3 - Article
C2 - 8265360
AN - SCOPUS:0027438369
SN - 0305-1048
VL - 21
SP - 5431
EP - 5438
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 23
ER -