Hübschmann T, Börner T, Hartmann E, Lamparter T
Humboldt Universität Berlin, Institut für Biologie/Genetik, Berlin, Germany. thomas=
Eur J Biochem. 2001 Apr;268(7):2055-63. doi: 10.1046/j.1432-1327.2001.02083.x.
The cph1 gene from the unicellular cyanobacterium Synechoycstis sp. PCC 6803 encodes a protein with the characteristics of plant phytochromes and histidine kinases of two-component phospho-relay systems. Spectral and biochemical properties of Cph1 have been intensely studied in vitro using protein from recombinant systems, but virtually nothing is known about the situation in the natural host. In the present study, His6-tagged Cph1 was isolated from Synechocystis cells. The cph1-his gene was expressed either under the control of the natural cph1 promoter or over-expressed using the strong promoter of the psbA2 gene. Upon purification with nickel affinity chromatography, the presence of Cph1 in extracts was confirmed by immunoblotting and Zn2+-induced fluorescence. The Cph1 extracts exhibited a red/far-red photoactivity characteristic of phytochromes. Difference spectra were identical with those of the phycocyanobilin adduct of recombinant Cph1, implying that phycocyanobilin is the chromophore of Cph1 in Synechocystis.
单细胞蓝藻集胞藻6803（Synechocystis sp. PCC 6803）的cph1基因编码一种具有植物光敏色素和双组分磷酸中继系统组氨酸激酶特征的蛋白质。利用重组系统中的蛋白质，已在体外对Cph1的光谱和生化特性进行了深入研究，但对于天然宿主中的情况几乎一无所知。在本研究中，从集胞藻细胞中分离出带有His6标签的Cph1。cph1-his基因要么在天然cph1启动子的控制下表达，要么使用psbA2基因的强启动子进行过表达。通过镍亲和层析纯化后，通过免疫印迹和Zn2+诱导荧光证实提取物中存在Cph1。Cph1提取物表现出光敏色素特有的红/远红光光活性。差异光谱与重组Cph1的藻蓝胆素加合物的光谱相同，这意味着藻蓝胆素是集胞藻中Cph1的发色团。
