ISOLATION, CHARACTERIZATION AND EXPRESSION OF THE GENE ENCODING CYTIDINE TRIPHOSPHATE SYNTHETASE FROM GIARDIA-INTESTINALIS

The cytidine triphosphate synthetase gene from Giardia intestinalis wa s cloned using a PCR-based strategy. A 519 bp PCR product was obtained from the amplification of genomic DNA using two oligonucleotides deri ved from the CTP synthetase amino acid consensus sequences DPYINVDPG a nd KTKPTQ. This product was used to probe restriction endonuclease dig ested genomic DNA and the respective plasmid mini-libraries. Two genom ic clones were obtained one with a 3.6 kb HindIII DNA fragment, contai ning approximately three-quarters of the 5'-end of the synthetase gene and subsequently, a 5.8 kb PstI DNA fragment which contained the whol e gene. The intronless gene has a 1863 bp open reading frame encoding 620 amino acids (M(r) of 68.3 kDa). A well conserved catalytic glutami ne aminotransferase (GAT) domain was identified. In addition, three in sert sequences were found which are not present in CTP synthetase from other species. Alignment and comparison of the deduced amino acid seq uence relative to CTP synthetases from other species revealed a high d egree of identity (34%) with a greater resemblance to prokaryotes than eukaryotes. The gene is located on chromosome 6 and the messenger RNA encoding it is estimated to be 1.9 kb. The coding region of G. intest inalis CTP synthetase was generated by PCR and subsequently cloned int o the pQE30 vector for expression in E. coli. This construct yielded a soluble and enzymatically active recombinant protein which was purifi ed by a Ni-NTA affinity column. The purified recombinant protein had a subunit molecular weight of 69.5 kDa and a native molecular weight of approximately 274 kDa. Kinetic studies of the partially purified reco mbinant G. intestinalis CTP synthetase gave apparent K-m values of 0.1 mM and approximately 0.5 mM for the substrates UTP and L-glutamine re spectively in accord with previously reported values for the native en zyme.