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刘冰冰, 陈煜, 邓明刚, 赵开弘, 周明. 嗜热藻BP-1中几个蓝细菌光敏色素结构域体内重组及光化学性质研究[J]. 植物科学学报, 2013, 31(2): 178-185. DOI: 10.3724/SP.J.1142.2013.20178
引用本文: 刘冰冰, 陈煜, 邓明刚, 赵开弘, 周明. 嗜热藻BP-1中几个蓝细菌光敏色素结构域体内重组及光化学性质研究[J]. 植物科学学报, 2013, 31(2): 178-185. DOI: 10.3724/SP.J.1142.2013.20178
LIU Bing-Bing, CHEN Yu, DENG Ming-Gang, ZHAO Kai-Hong, ZHOU Ming. Reconstitution in vivo and Photochemical Properties of Several Cyanobacteriochrome Domains from Thermosynechococcus elongatus BP-1[J]. Plant Science Journal, 2013, 31(2): 178-185. DOI: 10.3724/SP.J.1142.2013.20178
Citation: LIU Bing-Bing, CHEN Yu, DENG Ming-Gang, ZHAO Kai-Hong, ZHOU Ming. Reconstitution in vivo and Photochemical Properties of Several Cyanobacteriochrome Domains from Thermosynechococcus elongatus BP-1[J]. Plant Science Journal, 2013, 31(2): 178-185. DOI: 10.3724/SP.J.1142.2013.20178

嗜热藻BP-1中几个蓝细菌光敏色素结构域体内重组及光化学性质研究

Reconstitution in vivo and Photochemical Properties of Several Cyanobacteriochrome Domains from Thermosynechococcus elongatus BP-1

  • 摘要: 通过蛋白质序列同源性比对分析,在嗜热藻(Thermosynechococcus elongatus BP-1)里面找到了与已知的Pb/Pg型蓝细菌光敏色素TePixJ和TeTlr0924同源的3个基因tlr0911tlr1215tlr1999。通过分子克隆技术把它们的GAF结构域分别构建在pET30a(+)表达载体上,与可生成藻蓝胆素(PCB)的质粒pACYCDuet-ho1-pcyA在大肠杆菌BL21(DE3)体内重组,生成重组蛋白,利用亲和层析柱分离纯化,纯化后的蛋白质经过锌荧光和蛋白质酸性尿素变性以及荧光光谱和吸收光谱等实验分析鉴定,结果表明,Tlr0911-GAF存在蓝光吸收态Pb406 nm和绿光吸收态Pg527 nm之间的可逆光转换,它可共价结合两种藻胆色素,即藻紫胆素(PVB)和藻蓝胆素(PCB),Tlr1999-GAF则存在蓝光吸收态Pb417 nm和青光吸收态Pt496 nm之间的可逆光转换,它同样共价结合PVB和PCB, 而Tlr1215-GAF1和Tlr1215-GAF2不能自发结合藻胆色素,不具有光活性。

     

    Abstract: By protein sequence homology comparison with the Pb/Pg-type cyanobacteriochrome TePixJ and TeTlr0924, three homologous genes tlr0911, tlr1215 and tlr1999 from Thermosynechococcus elongatus BP-1 were found. By molecular cloning techniques, the GAF domains of those genes were cloned into expression vector pET30a(+), respectively. The E.coli strain BL21 (DE3) harboring the expression plasmid and the pACYCDuet-ho1-pcyA plasmid for phycocyanobilin (PCB) were induced to generate recombinant proteins. The expressed proteins (His)6 tagged at the N-terminus were purified with nickel-affinity His-Trap chelating column. The purified proteins were identified with zinc-induced fluorescence, acidic urea denaturation, fluorescence and absorption spectrum. Results showed that Tlr0911-GAF contained two covalently bound bilin chromophores, phycoviolobilin (PVB) and phycocyanobilin (PCB), and exhibited reversible photoconversion between a blue-absorbing form at 406 nm (Pb406 nm) and a green-absorbing form at 527 nm (Pg527 nm). Tlr1999-GAF was also covalently bound with PVB and PCB, and reversible photoconversion existed between a blue-absorbing form at 417 nm (Pb417 nm) and a teal-absorbing form at 496 nm (Pt496 nm). Neither Tlr1215-GAF1 nor Tlr1215-GAF2 could be spontaneously bound with bilin chromophore.

     

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