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
PHYCOBILIN COVALENT COUPLED TO β SUBUNIT CYS-155 OF PHYCOCYANIN AND PHYCOERYTHROCYANIN FROM MASTIGOCLADUS LAMINOSUS PCC 7603
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摘要: 层理鞭枝藻(Mastigocladus laminosus PCC7603)藻蓝蛋白β-CPC和藻红蓝蛋白β-PEC中均存在2个藻胆色素结合位点(Cys-84和Cys-155),可与藻蓝胆素(简称PCB)发生共价偶联反应,已有研究证实编码基因为alr0617的裂合酶CpcS1是催化Cys-84与PCB共价偶联的裂合酶。在研究Cys-155与PCB共价偶联的过程中,通过BLAST软件同源性对比分析后,筛选出4个基因:cpcT1、cpcT2、cpcS1、cpcS2,其中基因cpcT1和cpcS2,利用分子克隆的技术,根据实验需要转到载体pCDFDuet上,通过DNA电泳和蛋白质电泳挑选出正确的克隆。此4个基因对应的质粒与在大肠杆菌内生成PCB必需的质粒pACYCDuet-ho1-pcyA,以及质粒pET-cpcB(C84S)或pET-pecB(C84A),共同转入大肠杆菌BL21(DE3)内,进行体内重组,得到各重组蛋白,经过亲和层析柱提纯并透析,过滤掉金属离子,纯化透析后的蛋白经过活性比较、蛋白质电泳以及锌染色、蛋白质变性等试验以及荧光和紫外吸收光谱等鉴定,通过与相应文献中PCB光谱的比对,确定编码基因为all5339的裂合酶CpcT1能高效地催化Cys-155与PCB共价偶联,而其余3个基因不能起到催化作用。由此,能催化脱辅基蛋白β-CPC和β-PEC的两个位点共价偶联PCB的裂合酶均被发现。实验对于研究藻胆蛋白的生物合成、光合作用捕光机理以及藻胆体的组装等有重要的意义。
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关键词:
- 藻蓝蛋白β-CPC /
- 藻红蓝蛋白β-PEC /
- 裂合酶CpcT1 /
- 体内重组
Abstract: 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 the lyase of Cys-155, four genes were filtrated by BLAST comparison. They were cpcT1, cpcT2, cpcS1 and cpcS2, in which genes cpcT1 and cpcS2 had to be transformed to the vector pCDFDuet for the experiments need by molecular cloning technique, and the correct clones could be selected by agarose gel electrophoresis and SDS-PAGE. The entire pathway of a fluorescent β subunit was synthesized from combinational expression of four genes in three compatible pACYCDuet-ho1-pcyA, pET30a-cpcB (C84S) or pET30a-pecB (C84A) and one of the filtrated four genes in E. coli, in LB culture medium at 20 ℃ for 12h. After mechanical disruption, the reconstitution 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, SDS-PAGE, chromoprotein denaturation, absorbance and fluorescence spectrum. As a result, compared to the related literatures of PCB spectrum, 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’s two sites of β subunits and PCB were all found. This result will be signality for researching the phycobiliprotein biosynthesis, light-harvesting principle in photosynthesis, assembling phycobilisome and so on.-
Keywords:
- β-CPC /
- β-PEC /
- Lyase CpcT1 /
- Reconstitution in vivo
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