Isolation, sequencing and expression of the gene encoding hypoxanthine-guanine-xanthine phosphoribosyltransferase of Tritrichomonas foetus.
We have cloned and expressed the full-length gene encoding the hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from the anaerobic protozoan parasite Tritrichomonas foetus. This enzyme is essential in nucleic acid metabolism of T. foetus because the parasite is unable to synthesize purine nucleotides de novo and relies on the HGXPRTase activities for its purine requirements. Initially, a cDNA clone encoding part of the HGXPRTase was isolated by complementation of an Escherichia coli mutant, SO609, with a cDNA library of T. foetus. Northern blot analysis identified a single mRNA band of approximately 700-800 bases. The full-length genomic clone was then isolated and identified to have an open reading frame of 549 bp encoding an 183-amino acid 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 active recombinant T. foetus HGXPRTase, which constitutes approximately 20% of the total cellular protein of the transformed E. coli. It has the same molecular weight as the authentic native enzyme, and the N-terminal amino acid sequence of the recombinant enzyme is identical to that predicted from the open reading frame. The high expression of this apparently native T. foetus HGXPRTase will provide large quantities of purified protein, necessary for detailed kinetic and structural analysis of this enzyme for its potential value as a target for antitrichomonial chemotherapy. To our knowledge, this is also the first time a gene from T. foetus was cloned and expressed.
Chin
M S
MS
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Wang
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D017125:Genes, Protozoan / Q000235:genetics*; D005816:Genetic Complementation Test; D015698:Genomic Library; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011687:Purines / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D016835:Tritrichomonas foetus / Q000201:enzymology / Q000235:genetics*
D018076:DNA, Complementary; D011687:Purines; D011994:Recombinant Proteins; D010430:Pentosyltransferases; C083867:hypoxanthine-guanine-xanthine phosphoribosyltransferase