藻蓝蛋白裂合酶催化藻红胆素(PEB)与CpcA和PecA共价偶联  

Phycoerythrobilin covalent coupled to CpcA and PecA with phycocyanin lyase from Anabaena sp. PCC 7120

作　　者：张举成[1,2] 伍贤军[2] 张娟[3] 刘卫[1] 周明[3,4] 

机构地区：[1]红河学院理学院,云南蒙自661100 [2]华中科技大学环境科学与工程学院,湖北武汉430074 [3]华中农业大学农业微生物国家重点实验室,湖北武汉430070 [4]华中农业大学生物质与生物能源研究中心,湖北武汉430070

出　　处：《云南大学学报（自然科学版）》2009年第6期632-637,648,共7页Journal of Yunnan University(Natural Sciences Edition)

基　　金：国家自然科学基金资助项目(30870519);教育部高校骨干教师访学计划资助项目

摘　　要：将由鱼腥藻Anabaenasp.PCC 7120中cpcE/F构建的pCOLADuet-cpcE/F质粒和pACYCDuet-pebA质粒、pCDFDuet-hol-pebB质粒、pETDuet-pecA质粒、pETDuet-cpcA质粒转化入大肠杆菌,在IPTG的诱导下,实现了色素蛋白PEB-CpcA和PEB-PecA的成功表达.吸收光谱和荧光光谱研究表明,CpcE/F能够催化PEB共价连接到CpcA和PecA的Cys-84上形成色素蛋白PEB-PecA和PEB-CpcA,色素蛋白的最大吸收峰为555 nm,最强荧光峰为570 nm.Zn2+电泳分析结果表明,体内重组获得了目标色素蛋白,证明由cpcE/F所编码的蛋白裂合酶在体内重组中能够催化PEB与CpcA和PecA的共价偶联反应,生成非天然色素蛋白PEB-CpcA和PEB-PecA.The pCOLADuet-cpcE/F plasmid,which expressed the phycocyaninlyase encoded by cpcE/F in Anabaena sp.PCC 7120,and other plasmids,including pACYCDuet-pebA,pCDFDuet-hol-pebB,pETDuet-pecA or pETDuet-cpcA,were transformed together in E.coli BL21（DE3）.After being induced by IPTG,the chromoproteins PEB-CpcA and PEB-PecA were successfully expressed in E.coli.The Absorption and fluorescence spectra showed that CpcE/F could catalyse the covalent attachment of PEB at Cys-84 of α-subunit of phycocyanin and phycoerythrocyanin to form the chromoprotein PEB-PecA and PEB-CpcA,respectively.Themaximal Absorption of PEB-PecA and PEB-CpcA was 555 nm,and the maximal fluorescence was 570 nm.SDS-PAGE analysis displayed the Zn2＋-induced fluorescence of the bound chromophore under irradiation by 280 nm,which showed that PEB-PecA and PEB-CpcA were got with the chromophorylation in E.coli.The results showed that the lyase CpcE/F could catalyse the covalent attachment of PEB to CpcA and PecA.

关 键 词：藻蓝蛋白α-CPC 藻红蓝蛋白α-PEC 裂合酶CpcE/F 体内重组 藻红胆素 

分 类 号：Q946.1[生物学—植物学]