Expression of human Cu, Zn-superoxide dismutase in an insect cell-free system and its structural analysis by MALDI-TOF MS
2009
Ezure, Toru | Suzuki, Takashi | Ando, Eiji | Nishimura, Osamu | Tsunasawa, Susumu
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 Cu²⁺ and Zn²⁺ and was purified with a yield of approximately 33μg from 1ml 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.
显示更多 [+] 显示较少 [-]