蓝藻藻胆蛋白裂合酶cpcs和cpct的蛋白质工程分析-protein engineering analysis of cyanobacteria phycobiliprotein lyase cpcs and cpct.docxVIP

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蓝藻藻胆蛋白裂合酶cpcs和cpct的蛋白质工程分析-protein engineering analysis of cyanobacteria phycobiliprotein lyase cpcs and cpct.docx

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蓝藻藻胆蛋白裂合酶cpcs和cpct的蛋白质工程分析-protein engineering analysis of cyanobacteria phycobiliprotein lyase cpcs and cpct

AbstractPhycobiliproteins are light-harvesting proteins present in cyanobacteria. The most important step in phycobilin biosynthesis is the phycobilin addition to the apo-phycobiliproteins. By homology analyses, the protein encoded by gene alr0617 was proved to be the lyase of Cys-84 of ?-CPC, which named CpcS. And the protein encoded by gene all5339 was proved to be the lyase of Cys-155 of ?-CPC, which named CpcT.For next research, the conditions of CpcS purification are useful. In this laboratory,gene cpcS from Anabaena sp. PCC 7120 has been cloned to the vector pET30a(+), which can expressed the lyase CpcS fused 48 amino acids, including 6 consecutive histidines as his-tag. Construct the plasmid pCDFDuet-1-cpcS, expressing the his-tagged protein fused14 amino acids. The two fusion protein types can be purified via Ni2+-affinitychromatography on chelating Sepharose. And then futher purified via FPLC with a DEAE FF column. As a result, the purification conditions of the two types are the same. Use the buffer with imidazole up to 50mmol/L to elute the impurity sample, purifying via Ni2+-affinity chromatography. CpcS can be affinity-enriched after using the buffer containing 20mmol/L KPB, 0.3mol/L NaCl, pH6.5, when purified with a DEAE FF column.According to the conditions, purify some protein for research on the catalysis mechanism of CpcS. Via the reconstitution in vitro, the relative activity of CpcS(R18V) is 5% and CpcS(R147Q) is 13%. The percent of PCB bound of CpcS is 59%, CpcS(R18V) is 50%, and CpcS(R147Q) is 16%.CpcT resembles CpcS. For the research on the catalysis mechanism of CpcT, the mutants are necessary. Construct the gene mutants cpcT(H33F) and cpcT(R97A) via TaKaRa MutanBEST Kit. Then the protein mutants can be expressed.Keywords: phycocyanobilin; reconstitution in vitro; site-directed mutation; spectrum analysis目录摘要 IAbstract II目录III1序言1.1蓝藻及其藻胆体（1）1.2藻胆色素与藻胆蛋白（3）1.3藻胆蛋白光合作用能量的传递（5）1.4藻胆色素与藻胆蛋白连接机制的研究现状（7）1.5研究意义及展望（8）2鱼腥藻藻蓝蛋