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马琼, 向建伟, 牟若兰, 邓腊青, 王运, 周明. 蓝细菌光敏色素Alr1966GAF2及其突变体的表达与光化学性质研究[J]. 植物科学学报, 2020, 38(4): 551-557. DOI: 10.11913/PSJ.2095-0837.2020.40551
引用本文: 马琼, 向建伟, 牟若兰, 邓腊青, 王运, 周明. 蓝细菌光敏色素Alr1966GAF2及其突变体的表达与光化学性质研究[J]. 植物科学学报, 2020, 38(4): 551-557. DOI: 10.11913/PSJ.2095-0837.2020.40551
Ma Qiong, Xiang Jian-Wei, Mou Ruo-Lan, Deng La-Qing, Wang Yun, Zhou Ming. Expression and photochemical properties of cyanobacteriochrome Alr1966GAF2 and its mutants[J]. Plant Science Journal, 2020, 38(4): 551-557. DOI: 10.11913/PSJ.2095-0837.2020.40551
Citation: Ma Qiong, Xiang Jian-Wei, Mou Ruo-Lan, Deng La-Qing, Wang Yun, Zhou Ming. Expression and photochemical properties of cyanobacteriochrome Alr1966GAF2 and its mutants[J]. Plant Science Journal, 2020, 38(4): 551-557. DOI: 10.11913/PSJ.2095-0837.2020.40551

蓝细菌光敏色素Alr1966GAF2及其突变体的表达与光化学性质研究

Expression and photochemical properties of cyanobacteriochrome Alr1966GAF2 and its mutants

  • 摘要: 采用PCR技术从鱼腥藻(Anabaena sp.PCC7120)中扩增蓝细菌光敏色素基因片段alr1966gaf2,将alr1966gaf2插入到pET-30a(+)载体中,构建表达质粒pET-alr1966gaf2。最后将Alr1966GAF2与HO1、PcyA在E.coli BL21(DE3)中共表达获得色素蛋白Alr1966GAF2,并对该蛋白的光化学性质进行分析。结果显示,色素蛋白Alr1966GAF2结合色素为藻蓝胆素(phycoerythrobilin,PCB)或藻紫胆素(phycoviolobilin,PVB),在3种不同吸收态15Z-P428 nm、中间态和15E-P514 nm之间具有顺序可逆光效应。通过定点突变技术将DXCF基序中的保守性Cys突变为Ala,获得了突变体Alr1966GAF2(C72A)。将Alr1966GAF2(C72A)与HO1、PcyA共表达,获得色素蛋白Alr1966GAF2(C72A)。研究结果表明Alr1966GAF2(C72A)结合色素为PCB,Alr1966GAF2(C72A)-PCB具有较强的荧光活性,其荧光量子的产率高达0.11。Alr1966GAF2(C72A)不仅能够共价结合PCB,还可以结合胆绿素(Biliverdin,BV),均具有较强的红色荧光活性。

     

    Abstract: To construct pET-alr1966gaf2, a fragment of alr1966gaf2 was amplified by polymerase chain reaction (PCR) from Anabaena sp. PCC7120, and then inserted into pET-30a(+). For over-expression, pET-alr1966gaf2 was transformed into Escherichia coli BL21(DE3) containing pACYC-ho1-pcyA and biliproteins were co-expressed successfully. Results showed that bili-Alr1966GAF2 had a sequential reversible photoconversion in three different states. We also detected red fluorescence reversible photoconversion of Alr1966GAF2 in 15E-P514 nm/15Z-P428 nm forms. Via site-directed mutagenesis, we mutated conserved Cys into Ala in the conserved DXCF motif of Alr1966GAF2, resulting in Alr1966GAF2(C72A). Alr1966GAF2(C72A)-PCB showed strong red fluorescence and high fluorescence quantum yield of 0.11. Furthermore, Alr1966GAF2(C72A) could bind to phycoerythrobilin (PCB) and biliverdin (BV) covalently, with strong red fluorescence. Therefore, as a red fluorescent protein, Alr1966GAF2(C72A) could be further developed as a fluorescent probe and applied in life sciences.

     

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