Expression,purification and characterization of the soluble Cu_A domain of cytochrome c oxidase of Paracoccus versutus  

作　　者：LI Lianzhi SONG Aixin XIE Yi HUANG Zhongxian Ellen de Waal Urszula Kolczak Gerard W.Canters 

机构地区：[1]Chemical Biology Laboratory,Department of Chemistry,Fudan University,Shanghai 200433,China [2]State Key Laboratory of Genetics,School of Life Science,Fudan University,Shanghai 200433,China [3]Leiden Institute of Chemistry,Leiden University,PO Box 9502,2300 RA Leiden,The Netherlands

出　　处：《Chinese Science Bulletin》2001年第19期1608-1612,共5页

基　　金：This work was supported by the National Natural Science Foundation of China (Grant No. 39990600).

摘　　要：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-CuThe key subunit II of cytochrome c oxidase (CcO) contains a soluble binuclear copper center (CuA) domain. The Cua domain ofParacoccus versutus was cloned, expressed, purified and characterized. The gene encoding the Cua domain in pET11d vector was expressed inE. coli BL21 (DE3). The results showed that the Cua domain was expressed mostly in inclusion bodies and the Cua domain protein synthesized inE. coli cells represents approximately 10 percent of the total cellular proteins. Dissolved in urea, dialyzed 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 in the mixed-valence binuclear metal center (Cu2S2R2). The far-UV CD spectrum indicated that this domain is predominantly in β-sheet structure. The fluorescence spectra showed that its maximal excitation wavelength and maximal emission wavelength are at 280 and 345 nm, respectively.

关 键 词：CYTOCHROME coxidase CuA DOMAIN protein gene EXPRESSION PURIFICATION spectra. 

分 类 号：Q51[生物学—生物化学]