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.
Ezure
Toru
T
Clinical and Biotechnology Business Unit, Life Science Business Department, Analytical and Measuring Instruments Division, Nakagyo-ku, Kyoto 604-8511, Japan. ezure@shimadzu.co.jp
Suzuki
Takashi
T
Ando
Eiji
E
Nishimura
Osamu
O
Tsunasawa
Susumu
S
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