Rapid and discrete isolation of oxygen-evolving His-tagged photosystem II core complex from Chlamydomonas reinhardtii by Ni2+ affinity column chromatography.
We have developed a simple and rapid procedure to isolate an oxygen-evolving photosystem II (PS II) core complex from Chlamydomonas reinhardtii. A His-tag made of six consecutive histidine residues was genetically attached at the carboxy terminus of D2 protein to create a metal binding site on the PS II supramolecular complex. The recombinant cells producing the His-tagged variant of D2 protein grew photoautotrophically as well as the wild-type cells. Characterization of the oxygen evolution and the thermoluminescence properties revealed that the His-tagging did not affect the functional integrity of the PS II reaction center. A PS II core complex was isolated from the detergent-solubilized thylakoids of the recombinant cells in 4 h by a single one-step Ni2+ affinity column chromatography. This preparation consists of D1, D2, CP43, CP47, 33 kDa, and a few low molecular weight proteins, and retains a high rate of oxygen-evolving activity (= 1000 micromol/mg Chl/h).
Sugiura
M
M
Photosynthesis Research Laboratory, Institute of Physical and Chemical Research (RIKEN), Wako, Saitama, Japan. miwa@postman.riken.go.jp
Inoue
Y
Y
Minagawa
J
J
D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000737:chemistry*; D002846:Chromatography, Affinity; D006639:Histidine; D049449:Luminescence; D009532:Nickel; D010788:Photosynthesis; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry*; D045332:Photosystem II Protein Complex; D011994:Recombinant Proteins / Q000302:isolation & purification; D013057:Spectrum Analysis
D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D011994:Recombinant Proteins; D006639:Histidine; D009532:Nickel