Purification and stabilization of a monomeric isocitrate dehydrogenase from Corynebacterium glutamicum.
Monomeric isocitrate dehydrogenase was expressed in Corynebacterium glutamicum cells harboring pEK-icdES1, a plasmid carrying the gene for the enzyme. Two- to three-fold higher expression levels of the recombinant enzyme were observed in such cells when grown in fermentors, compared to those grown in shaker incubators. The enzyme was purified to homogeneity by ammonium sulfate fractionation, Sephadex G-150 gel filtration, FPLC Mono Q anion-exchange chromatography, and affinity gel chromatography. Approximately 4 mg of 98% pure recombinant enzyme was obtained per liter of bacterial culture. Our results also include optimum buffer conditions for purification and storage of the enzyme.
Bai
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C
College of Medicine, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, S7N 5E5, Canada.
Fernandez
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E
Yang
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H
Chen
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R
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003352:Corynebacterium / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D007521:Isocitrate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D007521:Isocitrate Dehydrogenase