Identification Of Phycobilin From Cyanobacteria In Vivo

To study the structure-function relationships and the effect of the amino acid to the CpeS of phycocyanin,site-directed mutations were used to generate four mutants, cpeS(C51T), cpeS(C63T), cpeS(R147Q) and cpeS(R149L). The six primers were designed, of which primer P1, P2 and P6 were used to generate mutant cpeS(C51T), primer P1, P3 and P6 were used to generate cpeS(C63T), primer P1, P4 and P6 were used to generate cpeS(R147Q), primer P1, P5 and P6 were used to generate cpeS(R149L). The PCR products were purified and digested with Smaâ… and Xhoâ… , then recombined with pBluescript vector and transfonned into the E. coli TG1 strains. It was confirmed that the genes were inserted into pBluescript vector by the restriction enzyme double digestion with Smaâ… and Xhoâ… , PCR and sequencing. The genes of the four amino acid mutants were purified after the Smaâ… and Xhoâ… double digestion of the reconstructed plasmids, and then inserted into expression vector pET 30a(+), finally transformed into E. coli BL21(DE3). In the transformed E. coli BL21(DE3) strains, about 25.9 kD protein were overexpressed, respectively.In order to study the effect of the amino acid to the CpeS,the cpeS(C51T), cpeS(C63T), cpeS(R147Q) and cpeS(R149L) was introduced into E. coli BL21(DE3) with cpcB(C155I), respectively, together with genes ho1 (encoding heme oxygenase) and pcyA(encoding PCB:ferredoxin oxidoreductase). The purified reconstituted products were determined with absorption spectra and fluorescence spectra, showing that the CpeS(C63T) and the CpeS(R147Q) has activity to link CpcB(C155I) with pigment PCB in vivo, while the CpeS(C51T) and the CpeS(R149L) has not.We also introduced the aphA(26-320) into E.coli together with genes ho1 (encoding heme oxygenase) and pcyA(encoding PCB:ferredoxin oxidoreductase) to realize the autoassembly of the Cyanobacterial phytochrome the AphA(26-320) and the PCB.