Rapid and discrete isolation of oxygen-evolving His-tagged photosystem II core complex from Chlamydomonas reinhardtii by Ni2+ affinity column chromatography
Rapid and discrete isolation of oxygen-evolving His-tagged photosystem II core complex from Chlamydomonas reinhardtii by Ni2+ affinity column chromatography
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DOI：
10.1016/s0014-5793(98)00328-7
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发表时间：
1998-04-10
期刊：
影响因子：
3.5
通讯作者：
Minagawa, J
中科院分区：
文献类型：
--
作者：
Sugiura, M;Inoue, Y;Minagawa, J
我们开发了一种简单快速的程序来从莱茵衣藻中分离出释氧光系统 II (PS II) 核心复合物。由sis连续组氨酸残基组成的His标签通过遗传方式附着在D2蛋白的羧基末端，以在PS II超分子复合物上产生金属结合位点。产生D2蛋白的His标签变体的重组细胞与野生型细胞一样光自养生长。析氧和热释光特性的表征表明，His 标记不会影响 PS II 反应中心的功能完整性。通过单一一步Ni 2+ 亲和柱层析，在4小时内从重组细胞的去污剂溶解的类囊体中分离出PS II核心复合物。该制剂由D1、D2、CP43、CP47、33 kDa和一些低分子量蛋白质组成，并保留高放氧活性率(=1000 mu mol/mg Chl/h)，(C) 1998欧洲生化学会联合会。
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 sis consecutive histidine residues was genetically attached at the carboxy terminus of D2 protein to create a metal binding site on the PS Il supramolecular complex, The recombinant cells producing the His-tagged variant of D2 protein grew photo-autotrophically 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 Il 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 mu mol/mg Chl/h), (C) 1998 Federation of European Biochemical Societies.