Expression and one-step purification of recombinant Arabidopsis thaliana frataxin homolog (AtFH).
Frataxin, a nuclear-encoded mitochondrial protein, has been proposed to participate in Fe-S cluster assembly, mitochondrial energy metabolism, respiration, and iron homeostasis. However, its precise function remains elusive. Frataxin is highly conserved in living organisms with no major structural changes, in particular at the C-terminal protein domain, suggesting that it plays a key function in all organisms. Recently, a plant gene, AtFH, with significant homology to other members of the frataxin family has been described. To gain insight on the frataxin role in plants, the frataxin domain was expressed in Escherichia coli BL21-codonPlus (DE3)-RIL cells and purified using a Ni-chelating column. The purified protein, added to a mixture containing Fe(II) and H2O2, attenuates the Fenton reaction indicating that the recombinant plant frataxin is functional. The procedure described here produced high yield of 99% pure protein through only one chromatographic step, suitable for further structure-function studies.
Maliandi
Maria V
MV
IIB-INTECH, UNSAM-CONICET, CC 164 (7130) Chascomús, Argentina.
Busi
Maria V
MV
Clemente
Marina
M
Zabaleta
Eduardo J
EJ
Araya
Alejandro
A
Gomez-Casati
Diego F
DF
D017360:Arabidopsis / Q000737:chemistry; D029681:Arabidopsis Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D033862:Iron-Binding Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D000096923:Frataxin
D029681:Arabidopsis Proteins; D006861:Hydrogen Peroxide; D007501:Iron; D033862:Iron-Binding Proteins; D024101:Mitochondrial Proteins; D000096923:Frataxin; C518592:FH protein, Arabidopsis; C045076:Fenton's reagent