The key subunit II of cytochrome c oxidase (CcO) contains a soluble binuclear copper center (CuA) domain. The CUA domain of Paracoccus versutus was cloned, expressed, purified and characterized. The gene encoding the ...The key subunit II of cytochrome c oxidase (CcO) contains a soluble binuclear copper center (CuA) domain. The CUA domain of Paracoccus versutus was cloned, expressed, purified and characterized. The gene encoding the CUA domain in pETlld vector was expressed in E. coli BL21 (DE3). The results showed that the CuA domain was expressed mostly in inclusion bodies and the CUA domain protein synthesized in E. coli cells represents approximately 10 percent of the total cellular proteins. Dissolved in urea, dia-lyzed and recombined with Cu+/Cu2+ and purified by the Q-sepharose fast flow anion-exchange column and Sephadex G-75 gel filtration column, the soluble purple-colored protein, which shows a single band in electrophoresis, was obtained. The UV-visible absorption spectrum of CUA domain showed that there are intense band at 478 nm and a shoulder peak at 530 nm, and two weak bands at 360 and 806 nm respectively, which can be assigned to the charge transfer and the interactions of obitals of Cu-S and Cu-Cu展开更多
基金This work was supported by the National Natural Science Foundation of China (Grant No. 39990600).
文摘The key subunit II of cytochrome c oxidase (CcO) contains a soluble binuclear copper center (CuA) domain. The CUA domain of Paracoccus versutus was cloned, expressed, purified and characterized. The gene encoding the CUA domain in pETlld vector was expressed in E. coli BL21 (DE3). The results showed that the CuA domain was expressed mostly in inclusion bodies and the CUA domain protein synthesized in E. coli cells represents approximately 10 percent of the total cellular proteins. Dissolved in urea, dia-lyzed and recombined with Cu+/Cu2+ and purified by the Q-sepharose fast flow anion-exchange column and Sephadex G-75 gel filtration column, the soluble purple-colored protein, which shows a single band in electrophoresis, was obtained. The UV-visible absorption spectrum of CUA domain showed that there are intense band at 478 nm and a shoulder peak at 530 nm, and two weak bands at 360 and 806 nm respectively, which can be assigned to the charge transfer and the interactions of obitals of Cu-S and Cu-Cu
