Characterisation and expression of the carbamate kinase gene from Giardia intestinalis.
The arginine dihydrolase pathway in Giardia intestinalis produces energy via the carbamate kinase (CBK, ATP:carbamate phosphotransferase, EC 2.7.2.2) reaction. Characterisation of the CBK gene from the Portland 1 strain indicated that it is located on either chromosome 3 or 4, does not appear to contain introns and is expressed in both the trophozoite and early cyst stages. Heterologous expression of CBK in Escherichia coli, using the pQE-30 expression system (QIAGEN), enabled a one-step purification of the recombinant enzyme via affinity chromatography. The expressed protein was identified by enzyme assay and mass spectrometry. The native and recombinant forms of the enzyme have similar physical properties and the recombinant enzyme appears to be active as the homodimer.
Minotto
L
L
School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, Australia.
Tutticci
E A
EA
Bagnara
A S
AS
Schofield
P J
PJ
Edwards
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004734:Energy Metabolism; D017125:Genes, Protozoan; D016829:Giardia lamblia / Q000201:enzymology / Q000235:genetics*; D006867:Hydrolases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011994:Recombinant Proteins; D017851:Phosphotransferases (Carboxyl Group Acceptor); C019288:carbamate kinase; D006867:Hydrolases; C019723:arginine deiminase