Heterologous expression of recombinant proteinsis an essential technology for protein characterization.A major obstacle to investigating the biochemicalproperties of membrane proteins is the difficulty inobtaining sufficient amounts of functional protein. Herewe report the successful expression of the tricarboxylate(or citrate) carrier (CIC) of eel (Anguilla anguilla) fromSpodoptera frugiperda (Sf9) cells using the baculovirusexpression system. The recombinant CIC was purified byaffinity chromatography on Ni2+-NTA agarose; the yieldof the purified active protein was 0.4–0.5 mg/l of culture.The transport characteristics of the recombinant CIC andthe effects of inhibitors on transport are similar to thosedetermined for eel liver mitochondrial CIC. Because theCIC is one member of an extensive family of mitochondrialtransport proteins, it is likely that the procedure usedin this study to express and purify this carrier can besuccessfully applied to other mitochondrial transportproteins, thus providing sufficient protein for functionalcharacterization.

Abundant expression and purification of biologically active mitochondrial citrate carrier in baculovirus-infected insect cells

MADEO M;CAPPELLO, Anna Rita;MAZZEO, GIANCARLO;MONTALTO A;DOLCE, Vincenza
2009

Abstract

Heterologous expression of recombinant proteinsis an essential technology for protein characterization.A major obstacle to investigating the biochemicalproperties of membrane proteins is the difficulty inobtaining sufficient amounts of functional protein. Herewe report the successful expression of the tricarboxylate(or citrate) carrier (CIC) of eel (Anguilla anguilla) fromSpodoptera frugiperda (Sf9) cells using the baculovirusexpression system. The recombinant CIC was purified byaffinity chromatography on Ni2+-NTA agarose; the yieldof the purified active protein was 0.4–0.5 mg/l of culture.The transport characteristics of the recombinant CIC andthe effects of inhibitors on transport are similar to thosedetermined for eel liver mitochondrial CIC. Because theCIC is one member of an extensive family of mitochondrialtransport proteins, it is likely that the procedure usedin this study to express and purify this carrier can besuccessfully applied to other mitochondrial transportproteins, thus providing sufficient protein for functionalcharacterization.
Mitochondria . Citrate carrier . Membrane protein . Detergents . V5/His-tag . Baculovirus expression
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11770/153316
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