Functional expression of the gene encoding cytidine triphosphate synthetase from Plasmodium falciparum which contains two novel sequences that are potential antimalarial targets.
CTP synthetase (E C 6.3.4.2 UTP: ammonia ligase (ADP-forming)) catalyses the formation of CTP from UTP and, in the human parasite Plasmodium falciparum, is the sole source of cytidine nucleotides. It is thus a potential chemotherapeutic target, especially as the gene sequence indicated that the encoded GAT-domain of the enzyme contains two extended peptide segments (42aa and 223aa as compared to the host enzyme). Here, we circumvent the codon usage problems associated with the high A/T content of the P. falciparum sequence, especially evident in sequences encoding the extra peptides, to successfully express active recombinant P. falciparum CTP synthetase using preferred E. coli codons. This partially synthetic gene produced recombinant enzyme, containing the additional segments, which was functionally assayed for activity in vitro. We also show the native enzyme contains the additional peptides using immunoblots with antibodies derived from the recombinant protein. Confocal microscopy, using antibodies to the recombinant protein, provided evidence that the enzyme is expressed in vivo. This establishes for the first time that P. falciparum contain active CTP synthetase and that this enzyme contains two novel insert sequences in the functional enzyme.
Yuan
Ping
P
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia. p-yuan@northwestern.edu
Hendriks
Edward F
EF
Fernandez
Harvey R
HR
O'Sullivan
William J
WJ
Stewart
Thomas S
TS
D000818:Animals; D000962:Antimalarials / Q000494:pharmacology; D001483:Base Sequence; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D003570:Cytidine Triphosphate / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007120:Immunochemistry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014544:Uridine Triphosphate / Q000378:metabolism
D000962:Antimalarials; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D003570:Cytidine Triphosphate; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase; D014544:Uridine Triphosphate