蓝细菌光敏色素Alr1966GAF2及其突变体的表达与光化学性质研究

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-P_{428 nm}、中间态和15E-P_{514 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。

Expression and photochemical properties of cyanobacteriochrome Alr1966GAF2 and its mutants

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-P_{514 nm}/15Z-P_{428 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.