Bricker T M, Morvant J, Masri N, Sutton H M, Frankel L K
Department of Biological Sciences, Biochemistry and Molecular Biology Section, Louisiana State University, Baton Rouge, LA 70803, USA.
Biochim Biophys Acta. 1998 Nov 2;1409(1):50-7. doi: 10.1016/s0005-2728(98)00148-0.
Site-directed mutagenesis was used to produce a Synechocystis mutant containing a histidine tag at the C terminus of the CP 47 protein of Photosystem II. This mutant cell line, designated HT-3, exhibited slightly above normal rates of oxygen evolution and appeared to accumulate somewhat more Photosystem II reaction centers than a control strain. A rapidly isolatable (<7 h) oxygen-evolving Photosystem II preparation was prepared from HT-3 using dodecyl-beta-d-maltoside solubilization and Co2+ metal affinity chromatography. This histidine-tagged Photosystem II preparation stably evolved oxygen at a high rate (2440 micromol O2 (mg chl)-1 h-1), exhibited an alpha-band absorption maximum at 674 nm, and was highly enriched in a number of Photosystem II components including cytochrome c550. Fluorescence yield analysis using water or hydroxylamine as an electron donor to the Photosystem II preparation indicated that virtually all of the Photosystem II reaction centers were capable of evolving oxygen. Proteins associated with Photosystem II were highly enriched in this preparation. 3,3',5, 5'-Tetramethylbenzidine staining indicated that the histidine-tagged preparation was enriched in cytochromes c550 and b559 and depleted of cytochrome f. This result was confirmed by optical difference spectroscopy. This histidine-tagged Photosystem II preparation may be very useful for the isolation of Photosystem II preparations from mutants containing lesions in other Photosystem II proteins.
利用定点诱变技术构建了一种集胞藻突变体，该突变体在光系统II的CP 47蛋白的C末端含有组氨酸标签。这种突变细胞系命名为HT-3，其放氧速率略高于正常水平，且似乎比对照菌株积累了更多的光系统II反应中心。使用十二烷基-β-D-麦芽糖苷增溶和Co2+金属亲和色谱法，从HT-3中制备了一种可快速分离（<7小时）的放氧光系统II制剂。这种带有组氨酸标签的光系统II制剂能够以高速率稳定放氧（2440 μmol O2（mg chl）-1 h-1），在674 nm处呈现α带吸收最大值，并且高度富集了包括细胞色素c550在内的多种光系统II组分。使用水或羟胺作为光系统II制剂的电子供体进行荧光产率分析表明，几乎所有的光系统II反应中心都能够放氧。与光系统II相关的蛋白质在该制剂中高度富集。3,3',5,5'-四甲基联苯胺染色表明，带有组氨酸标签的制剂中细胞色素c550和b559富集，而细胞色素f缺失。这一结果通过光学差光谱法得到证实。这种带有组氨酸标签的光系统II制剂可能对于从其他光系统II蛋白存在损伤的突变体中分离光系统II制剂非常有用。
