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-ev olving photosystem II (PS II) core complex from Chlamydomonas reinhard tii. A His-tag made of sis consecutive histidine residues was genetica lly attached at the carboxy terminus of D2 protein to create a metal b inding site on the PS Il supramolecular complex, The recombinant cells producing the His-tagged variant of D2 protein grew photo-autotrophic ally as well as the wild-type cells. Characterization of the oxygen ev olution and the thermoluminescence properties revealed that the His-ta gging did not affect the functional integrity of the PS II reaction ce nter. A PS Il core complex was isolated from the detergent-solubilized thylakoids of the recombinant cells in 4 h by a single one-step Ni2affinity column chromatography. This preparation consists of D1, D2, C P43, CP47, 33 kDa, and a few low molecular weight proteins, and retain s a high rate of oxygen-evolving activity (=1000 mu mol/mg Chl/h), (C) 1998 Federation of European Biochemical Societies.