ISOLATION, SEQUENCING AND EXPRESSION OF THE GENE ENCODING HYPOXANTHINE-GUANINE-XANTHINE PHOSPHORIBOSYLTRANSFERASE OF TRITRICHOMONAS-FETUS

We have cloned and expressed the full-length gene encoding the hypoxan thine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from the anaerobic protozoan parasite Tritrichomonas foetus. This enzyme is ess ential in nucleic acid metabolism of T.foetus because the parasite is unable to synthesize purine nucleotides de novo and relies on the HGXP RTase activities for its purine requirements. Initially, a cDNA clone encoding part of the HGXPRTase was isolated by complementation of an E scherichia coil mutant, S phi 609, with a cDNA library of T. foetus. N orthern blot analysis identified a single mRNA band of approximately 7 00-800 bases. The full-length genomic clone was then isolated and iden tified to have an open reading frame of 549 bp encoding an 183-amino a cid sequence with an estimated size of 21.1 kDa. The sequence is only 27.3% identical to that of the human HGPRTase. The T. foetus HGXPRTase gene was subsequently cloned into the pBAce vector for expression in E. coli. This construct yields completely soluble and enzymatically ac tive recombinant T.foetus HGXPRTase, which constitutes approximately 2 0% of the total cellular protein of the transformed E. coli. It has th e same molecular weight as the authentic native enzyme, and the N-term inal amino acid sequence of the recombinant enzyme is identical to tha t predicted from the open reading frame. The high expression of this a pparently native T. foetus HGXPRTase will provide large quantities of purified protein, necessary for detailed kinetic and structural analys is of this enzyme for its potential value as a target for antitrichomo nial chemotherapy. To our knowledge, this is also the first time a gen e from T. foetus was cloned and expressed.