Expression of human Cu, Zn-superoxide dismutase in an insect cell-free system and its structural analysis by MALDI-TOF MS.
Human Cu, Zn-superoxide dismutase (hSOD1) is a homodimer that coordinates one copper and one zinc ion per monomer. These metal ions contribute to its enzymatic activity and structural stability. In addition, hSOD1 maintains an intra-subunit disulfide bond formed in the reducing environment of the cytosol and is active under a variety of stringent denaturing conditions. We report the expression of hSOD1 in a cell-free protein synthesis system constructed from Spodoptera frugiperda 21 (Sf21) insect cells, and its structural analysis including the status of the sole intra-subunit disulfide bond by mass spectrometry. By using this system hSOD1 was obtained in a soluble active form after addition of Cu(2+) and Zn(2+) and was purified with a yield of approximately 33 microg from 1 ml of reaction volume. Both enzymatic and structural analyses of the recombinant hSOD1 indicate that it was completely identical to the protein isolated from human erythrocytes.
Clinical and Biotechnology Business Unit, Life Science Business Department, Analytical and Measuring Instruments Division, Nakagyo-ku, Kyoto 604-8511, Japan. ezure@shimadzu.co.jp
Toru
T
Ezure
Takashi
T
Suzuki
Eiji
E
Ando
Osamu
O
Nishimura
Susumu
S
Tsunasawa
D000595:Amino Acid Sequence; D000818:Animals; D001709:Biotechnology / Q000379:methods; D002474:Cell-Free System / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera / Q000166:cytology; D013329:Structure-Activity Relationship; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism*
D013482:Superoxide Dismutase