细菌光敏色素AphA的克隆表达及重组新方法

Expression of cyanobacterial phytochrome AphA and its vitro reconstitution

通过PCR技术,从蓝藻PCC7120中扩增出细菌光敏色素AphA的基因aphA,进而构建aphA的缺失突变体aphA(N25/C445).二者分别转入表达载体pET30a中,在宿主菌中获得高效表达,获得的脱辅基蛋白与CpcA进行体外重组.在本体外重组体系中,以藻蓝蛋白α亚基(CpcA),在光敏色素自身作用或者藻蓝蛋白裂合酶的帮助下从CpcA上捕获胆色素完成重组.藻蓝蛋白裂合酶CpcE/F能很大程度增强光敏色素脱辅基蛋白从CpcA夺取PCB的能力.该结果对于研究细菌光敏色素的色素来源及生理生化机理都具有重要意义.

Anabaena sp. PCC 7120 cyanobacterial phytochrome gene AphA and its truncated mutant aphA (N-25/C-445) were PCR amplified and cloned. These DNA fragments could be expressed via vector peT30a in E. coli, respectively. The expressed apoproteins AphA and AphA (N-25/C-445) could be used for in vitro reconstitution of the cyanobacterial phytochrome. After the photoactivity of the reversible photochemistry of the reconstituted cyanobacterial phytochrome and it mutant was measured, it is seen that both AphA and AphA (N-25/C-445) can capture PCB from phycocyanin α subunit (CpcA), which can be speeded up efficiently with the phycocyanobilin lyase CpcE/F. This result is significant for the study on the biosynthesis of cyanobacterial phytochrome, especially the origin of its prosthetic chromophore.