Isolation, characterization and expression of the gene encoding cytidine triphosphate synthetase from Giardia intestinalis.
The cytidine triphosphate synthetase gene from Giardia intestinalis was cloned using a PCR-based strategy. A 519 bp PCR product was obtained from the amplification of genomic DNA using two oligonucleotides derived from the CTP synthetase amino acid consensus sequences DPYINVDPG and KTKPTQ. This product was used to probe restriction endonuclease digested genomic DNA and the respective plasmid mini-libraries. Two genomic clones were obtained one with a 3.6 kb HindIII DNA fragment, containing approximately three-quarters of the 5'-end of the synthetase gene and subsequently, a 5.8 kb PstI DNA fragment which contained the whole 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 glutamine aminotransferase (GAT) domain was identified. In addition, three insert sequences were found which are not present in CTP synthetase from other species. Alignment and comparison of the deduced amino acid sequence relative to CTP synthetases from other species revealed a high degree 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. intestinalis CTP synthetase was generated by PCR and subsequently cloned into the pQE30 vector for expression in E. coli. This construct yielded a soluble and enzymatically active recombinant protein which was purified 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 recombinant 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 respectively in accord with previously reported values for the native enzyme.
Lim
R L
RL
School of Biochemistry and Molecular Genetics, Faculty of Biological and Behavioural Sciences, University of New South Wales, Sydney, Australia.
O'Sullivan
W J
WJ
Stewart
T S
TS
D000595:Amino Acid Sequence; D000818:Animals; D019731:Carbon-Nitrogen Ligases; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D015870:Gene Expression; D017125:Genes, Protozoan; D016829:Giardia lamblia / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008025:Ligases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D017931:DNA Primers; D016054:DNA, Protozoan; D011994:Recombinant Proteins; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase