Purification and characterization of recombinant human farnesyl diphosphate synthase expressed in Escherichia coli.
We previously reported the isolation of a partial-length human fetal-liver cDNA encoding farnesyl diphosphate (FPP) synthase (EC 2.5.1.10) and the expression of an active FPP synthase fusion protein in Escherichia coli. The expressed human FPP synthase fusion protein has now been purified to apparent homogeneity by using two chromatographic steps. The purification scheme allowed the preparation of 1.8 mg of homogeneous protein from 149 mg of crude extract in a 64% yield with a 52-fold enrichment. A single band with a subunit molecular mass of 39 kDa was observed by Coomassie Blue staining after SDS/PAGE. A molecular mass of 78-80 kDa was calculated for the native form of the fusion protein by h.p.l.c. on a SEC-250 column, suggesting that the active fusion protein is a dimer. The purified fusion protein has FPP synthase condensation activities in the presence of both substrates, isopentenyl diphosphate and geranyl diphosphate. Enzyme activity was inhibited by a bisubstrate analogue of isopentenyl diphosphate and dimethylallyl diphosphate, and a small amount of higher prenyltransferase was observed. Michaelis constants for isopentenyl diphosphate and geranyl diphosphate were 0.55 and 0.43 microM respectively, and Vmax for synthesis of farnesyl diphosphate from these substrates was 1.08 mumol/min per mg. These results suggest that the structure and catalytic properties of the expressed FPP synthase fusion protein are virtually identical with those of the native human liver enzyme.
Ding
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Department of Biochemical Regulation, Merck Sharp & Dohme Research Laboratories, Rahway, NJ 07065.
Sheares
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Bergstrom
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Ponpipom
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Perez
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Poulter
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D019883:Alkyl and Aryl Transferases; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D051228:Geranyltranstransferase; D045782:Hemiterpenes; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D009943:Organophosphorus Compounds / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D014166:Transferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011106:Polyisoprenyl Phosphates; D011993:Recombinant Fusion Proteins; C004809:isopentenyl pyrophosphate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase