Characterization of wild-type and amyotrophic lateral sclerosis-related mutant Cu,Zn-superoxide dismutases overproduced in baculovirus-infected insect cells.
We describe the use of a baculovirus expression system to overproduce human Cu,Zn-superoxide dismutase (SOD). Spodoptera frugiperda (Sf21) insect cells infected with a baculovirus carrying the Cu,Zn-SOD cDNA synthesized a large amount of Cu,Zn-SOD apoprotein in the conventional medium. The SOD activity of the apoprotein, which was initially very low, increased in a dose-dependent manner when Cu2+ and Zn2+ were added to the medium. Cells grown in media supplemented with Cu2+ alone exhibited nearly maximal SOD activity. SOD activity reached 40% of the maximal level within 2 h after addition of Cu2+ to postinfected cells cultivated for 3 days in the conventional medium, and the activity gradually increased thereafter. The protein produced by the infected cells was purified by a simple procedure involving two chromatographic steps, DE52 ion exchange and ACA54 gel filtration. Identification of the recombinant Cu,Zn-SOD with the human erythrocyte enzyme was confirmed by immunochemical reactivity to anti-human Cu,Zn-SOD antibody and by partial amino acid sequencing of peptides from purified protein (50 amino acid residues in total). We constructed three mutant enzymes, which have been found in familial amyotrophic lateral sclerosis and are overproduced in Sf21 cells, and purified them. Mutant enzymes Gly41Asp, His43Arg, and Gly85Arg exhibited 47, 66, and 99% of wild-type SOD activity, respectively. The availability of this protein will facilitate investigation of the relationship between the structure and function of the mutant enzymes found in familial amyotrophic lateral sclerosis.
Fujii
J
J
Department of Biochemistry, Osaka University Medical School, Japan.
Myint
T
T
Seo
H G
HG
Kayanoki
Y
Y
Ikeda
Y
Y
Taniguchi
N
N
D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology*; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D001424:Bacterial Infections / Q000378:metabolism* / Q000473:pathology; D016367:Baculoviridae; D001483:Base Sequence; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003300:Copper / Q000494:pharmacology; D007477:Ions; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D018411:Spodoptera / Q000166:cytology; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
D001051:Apoenzymes; D007477:Ions; D015335:Molecular Probes; D003300:Copper; D013482:Superoxide Dismutase