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Rapid and discrete isolation of oxygen‐evolving His‐tagged photosystem II core complex from Chlamydomonas reinhardtii by Ni 2+ affinity column chromatography
Author(s) -
Sugiura Miwa,
Inoue Yorinao,
Minagawa Jun
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)00328-7
Subject(s) - chlamydomonas reinhardtii , photosystem ii , oxygen evolution , histidine , chemistry , affinity chromatography , recombinant dna , thylakoid , chlamydomonas , column chromatography , biophysics , biochemistry , chromatography , photosynthesis , biology , chloroplast , enzyme , electrode , mutant , electrochemistry , gene
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 Ni 2+ 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 μmol/mg Chl/h).