CLONING AND EXPRESSION OF THE HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE GENE FROM TRYPANOSOMA-BRUCEI

The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme of T rypanosoma brucei and related parasites provides a rational target tor the treatment of African sleeping sickness and several other parasiti c diseases. To characterize the T. brucei HGPRT enzyme in detail, the T. brucei hgprt was isolated within a 4.2 kb San-KpnI genomic insert a nd sequenced. Nucleotide sequence analysis revealed an open reading fr ame of 630 bp that encoded a protein of 210 amino acids with a M(r) = 23.4 kd. After gap alignment, the T. brucei HGPRT exhibited 21 - 23% a mino acid sequence identity, mostly in three clustered regions, with t he HGPRTs from human, S. mansoni, and P. falciparum, indicating that t he trypanosome enzyme was the most divergent of the group. Surprisingl y, the T. brucei HGPRT was more homologous to the hypoxanthine phospho ribosyltransferase (HPRT) from the prokaryote V. harveyi than to the e ukaryotic HGPRTs. Northern blot analysis revealed two trypanosome tran scripts of 1.4 and 1.9 kb, each expressed to equivalent degrees in ins ect vector and mammalian forms of the parasite. The T. brucei hgprt wa s inserted into an expression plasmid and transformed into Sphi606 E. coli that are deficient in both HPRT and xanthine-guanine phosphoribos yltransferase 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 enzy me recognized hypoxanthine, guanine, and allopurinol, but not xanthine or adenine, as substrates and was inhibited by a variety of nucleotid e effectors. The availability of a molecular clone encoding the T. bru cei hgprt and large quantities of homogeneous recombinant HGPRT enzyme provides an experimentally manipulable molecular and biochemical syst em for the rational design of novel therapeutic agents for the treatme nt of African sleeping sickness and other diseases of parasitic origin .