The Research Of GAF Domain In Cyanobacteriochromes AphC And RGS

Cyanobacteriochrome is a special type of phytochromes and phytochrome-related proteins which have been identified in cyanobacteria.The gaf1and gaf2of aphC (gene of PCC7120) are isolated from genomic DNA by PCR using specific primers and DNA polymerase. The two fragments, respectively, are subcloned into pET30-a (+). They respectively co-expressed with the plasmid inserted by heme oxygenase ho1and biliverdin reductase pcyA in E. coli. PCB-GAF1has been experimentally proved to show reversible photoconversion between15Z form and15E form upon irradiation with700nm light and570nm light. Denatured by acidic urea, the peaks of UV absorption are all at around662nm. It is indicated that PCB is attached to GAF1. The purified PCB-GAF1is detected via SDS-PAGE. After stained by Zn2+, it shows fluorescence under UV light, which is characteristic for bilins covalently bound to proteins. The molar extinction coefficient and fluorescence quantum yield can be obtained by using the data of the fluorescence and absorption spectra. Spectroscopy and zinc electrophoresis showed that GAF2can not connect to PCB. As a genetic encoding fluorescent probe like GFP family, chromoprotein PCB-GAF1has important application value in the biomedical label.Researchers in our laboratory have constructed a fluorescent protein based on cyanobacteriochrome GAF3domain from RGS of PCC6803, called GAF3:Ho1:PcyA. It can emit a strong fluorescence after expression in E. coli. On this basis, I use the fluorescent protein to locate MinD which is cell division protein. MinD is fused in the N-terminus of the fluorescent protein by molecular cloning techniques. By E. coli expression system, cells emit a maximum of670nm fluorescence, and fluorescence microscope imaging shows that MinD is locate at the ends of cell, which suggests the distribution of MinD in E. coli. Biliprotein GAF3:Ho1:PcyA is a very valuable fluorescent probe.