EXPRESSION AND PURIFICATION OF THE Chlamydomonas BLUE-COPPER PLASTOCYANIN PROTEIN

Electron transport;Copper proteins;Chlamydomonas;Plastocyanin

Ranatunga, Don RuwanKallas, Toivo ;

University of Wisconsin

关键词: Electron transport;    Copper proteins;    Chlamydomonas;    Plastocyanin;   

Others  :  https://minds.wisconsin.edu/bitstream/handle/1793/46732/Ranatunga%2c_Don_Thesis_%28With_TP%29._pdf.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: University of Wisconsin

【 摘 要 】

Plastocyanin (PC) is a type 1 blue copper (Cu) protein found in the thylakoidlumen of plant and algal chloroplasts and in some cyanobacteria. It carries electrons fromthe cytochrome bf complex to the photosystem I reaction center. The ?-barrel structure ofPC provides a binding site for the bound copper atom, which undergoes oxidation andreduction during electron transfer. In the oxidized state, the Cu center has a largeabsorbance peak centered at 597 nm, providing a convenient marker for purification andquantification of the protein. The atomic 3D structures of the Chlamydomonas reinhardtiicytochrome bf complex and PC have been solved. An abundant source ofChlamydomonas PC would be useful for detailed structure-function studies of electrontransfer and interactions between these important electron transfer partners. I haveexpressed in Escherichia coli the Chlamydomonas PC as a His-tagged, thioredoxin fusionprotein. The fusion protein, purified by metal-chelating and anion-exchangechromatography, carries a Cu atom as shown by its absorbance spectrum and has acharacteristic redox midpoint potential of +375.5 + 1.5 mV at pH 7.0. However, themolar Cu content of the purified protein was only ~10%. One hypothesis for low Cuincorporation is that differences in cis-trans isomerization of proline 36 (in mature C.reinhardti PC) near the Cu pocket might hinder Cu binding. Pro36 is in the less commoncis configuration in PC structures. This has been tested by site-directed mutations F35Sand AGF33-35KLS that made the PC protein of the green alga Chlamydomonas moresimilar to that of the cyanobacterium Synechocystis sp. PCC 6803. Unfortunately, thesemodifications did not significantly improve Cu incorporation, nor did attempts at in vitroCu reconstitution into the fusion protein nor the separated PC. Other possibilities for lowCu incorporation include interfering metal ion interactions during His-bind, Ni-chelatingchromatography or steric constraints imposed by the thioredoxin fusion partner. Anotherapproach for producing Chlamydomonas PC was the expression from plasmids thatencode the pelB leader sequence for export of the native PC to the E. coli periplasm.Although the PC yield from these plasmids is low, this strategy might avoid problemsthat appear to be associated with the thioredoxin fusion protein and immobilized metalion affinity chromatography.