Molecular Cloning, Expression and Purification of Arabidopsis thaliana Frataxin homolog (AtFH)

MALIANDI, M. V.; VALDEZ, H.; CLEMENTE, M.; BUSI, M. V.; EDUARDO ZABALETA; ARAYA, A.; DIEGO FABIAN GOMEZ CASATI

Pinamar

Congreso; XLI Reunión Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2005

It has been proposed that frataxin, a nuclear encoded mitochondrial protein, participates in Fe-S cluster assembly, mitochondrial energy metabolism, respiration and iron homeostasis, but its precise function remains elusive. This protein is highly conserved from bacteria to mammals and plants without major structural changes, specially at the C-terminal domain, suggesting that it could play similar function in all these organisms. We recently described AtFH, a plant gene with significant homology to other members of the frataxin family. The plant frataxin domain was expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL cells and purified by one-step Ni-chelating chromatography. The protein was determined to be 98% pure and migrates as a single protein band of about 15 kDa as assessed by SDS-PAGE. Arabidopsis null mutants deficient in AtFH expression showed higher rate of CO2 fixation respect to wt. Results also showed that this protein is escential for full-activity of succinate dehydrogenase (SDH) and aconitase, two mitochondrial iron-sulphur containing enzymes. Results are in agreement with the involvement of AtFH in Fe-S cluster assembly in plant mitochondria.