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

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.