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) i nsect cells infected with a baculovirus carrying the Cu,Zn-SOD cDNA sy nthesized a large amount of Cu,Zn-SOD apoprotein in the conventional m edium. The SOD activity of the apoprotein, which was initially very lo w, increased in a dose-dependent manner when Cu2+ and Zn2+ were added to the medium. Cells grown in media supplemented with Cu2+ alone exhib ited nearly maximal SOD activity. SOD activity reached 40% of the maxi mal level within 2 h after addition of Cu2+ to postinfected cells cult ivated for 3 days in the conventional medium, and the activity gradual ly increased thereafter. The protein produced by the infected cells wa s purified by a simple procedure involving two chromatographic steps, DE52 ion exchange and ACA54 gel filtration. Identification of the reco mbinant Cu,Zn-SOD with the human erythrocyte enzyme was confirmed by i mmunochemical reactivity to anti-human Cu,Zn-SOD antibody and by parti al amino acid sequencing of peptides from purified protein (50 amino a cid residues in total). We constructed three mutant enzymes, which hav e been found in familial amyotrophic lateral sclerosis and are overpro duced in Sf21 cells, and purified them. Mutant enzymes Gly(41)Asp, His (43)Arg, and Gly(85)Arg exhibited 47, 66, and 99% of wild-type SOD act ivity, respectively. The availability of this protein will facilitate investigation of the relationship between the structure and function o f the mutant enzymes found in familial amyotrophic lateral sclerosis.