Improving the purification of NAD+-dependent formate dehydrogenase from Candida methylica.
The Candida methylica (cm) recombinant wild type formate dehydrogenase (FDH) gene has been cloned into the pQE-2 TAGZyme expression vector and the 6xHis-tagged FDH gene has been overexpressed in JM105 cells to purify the FDH protein more efficiently, by the use of exopeptidases, TAGZyme Purification System, which has allowed the complete removal of the small N-terminal His-tag. After the purification procedure, 1.2 mg/mL cmFDH protein of >95% purity was obtained. The kinetic parameters of cmFDH have been determined by observing the oxidation of the nicotinamide coenzyme at 340 nm. The results have also been compared to the yield of standard vs. affinity purification of FDH.
Ordu
Emel Biçakçi
EB
Faculty of Science and Letters, Department of Molecular Biology and Genetics, Istanbul Technical University, Istanbul, Turkey.
Karagüler
Nevin Gül
NG
D001709:Biotechnology / Q000379:methods*; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D005560:Formate Dehydrogenases / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005800:Genes, Fungal; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D011994:Recombinant Proteins; D009243:NAD; D006639:Histidine; D005560:Formate Dehydrogenases