层理鞭枝藻藻蓝蛋白和藻红蓝蛋白β亚基Cys-155的藻胆色素共价偶联

Phycobiliproteins, as a homologous family of light-harvesting proteins present in cyanobacteria, red algae, and cryptophytes, are the function composition of light-harvesting complexes in the algae photosynthesis. The phycobiliprotein biosynthesis is the phycobilin addition to the apophycobiliproteins. So far, the correct attachments of most chromophores are catalyzed by lyases, of which only few have been characterized in vivo. In Mastigocladus laminosus PCC7603, phycocyanin and phycoerythrocyanin have two subunits, the coupling sites of phycobilin addition to the apophycobiliprotein of β subunits are Cys-84 and Cys-155. The protein CpcS1 encoded by gene alr0617 was proved to be the lyase of Cys-84. To search for the lyase of Cys-155, four genes were selected by BLAST comparison. They were cpcT1, cpcT2, cpcS1 and cpcS2, in which genes cpcT1 and cpcS2 had to be cloned to the vector pCDFDuet for the reconstitution experiments. And the correct clones could be determined by agarose gel electrophoresis and SDS-PAGE. The entire pathway of a fluorescent β subunit was reconstituted from combinational expression of four genes in three compatible pACYCDuet-ho1-pcyA, pET30a-cpcB (C84S) or pET30a-pecB (C84A) and one of the lyase genes in E. coli. The reconstituted protein were purified by metal chelating affinity chromatography, and dialysed in buffer overnight to leach the medical irons. At last, they were confirmed by lyase activity comparisons, absorbance and fluorescence spectra. As a result, the protein CpcT1 encoded by gene all5339 was the lyase of Cys-155. At the same time, it proved that the other three genes have no effects to the Cys-155. Up to now, the two lyases which could catalyze the coupled reaction of the apophycobiliprotein\\\