Expression, purification, and characterization of a bacterial GTP-dependent PEP carboxykinase.
The Corynebacterium glutamicum (C. glutamicum) phosphoenolpyruvate carboxykinase (PCK) gene (pckA) was cloned into an Escherichia coli expression vector with a glutathione S-transferase (GST) tag. This recombinant DNA can produce highly overexpressed tagged protein in soluble form. This is the first report of the production of C. glutamicum PCK overexpressed in E. coli. The GST-fused PCK was purified using the glutathione-Sepharose 4B affinity column and the GST tag was removed in one-step. This one-step, easy purification method would be very useful for future mutational and structural studies. The molecular mass of the purified protein is approximately 68 kDa as confirmed by mass spectrometry and it is a monomeric enzyme. Also, the enzyme assays revealed that C. glutamicum PCK has a GTP-specific activity and that its activity is maximal in the presence of both Mn2+ and Mg2+.
Aich
Sanjukta
S
Department of Biochemistry, University of Saskatchewan, Saskatoon, Sask, Canada S7N 5E5.
Imabayashi
Fumie
F
Delbaere
Louis T J
LT
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D016415:Sequence Alignment; D013379:Substrate Specificity
D001426:Bacterial Proteins; D008670:Metals; D010729:Phosphoenolpyruvate Carboxykinase (GTP)