集胞藻PCC6803中S2P蛋白酶假定底物的体外诱导表达及纯化
作 者：
秦春燕;陈谷
单 位：
华南理工大学轻工与食品学院
关键词：
第二位点蛋白酶(S2P);底物;anti-σ因子;原核表达;纯化
摘 要：
金属蛋白酶S2P同源蛋白在行光合作用的蓝藻中广泛存在.S2P蛋白酶通过在膜切割转录调控因子(anti-σ因子),释放σ因子来参与胁迫响应是跨膜信号转导的保守机制.集胞藻PCC6803中S2P蛋白酶参与胁迫响应的跨膜信号转导,但其作用机理及底物均未明确.经过深入考察分析,实验锁定了4对σ因子和anti-σ因子的组合:SigE-ChlH(Slr1055)、SigI(Sll0687)-Sll0688、SigG(Slr1545)-Slr1546及SigH(Slr0856)-Sll0857.以pET-30b(+)为载体,通过重组表达纯化,成功获得一系列S2P假定底物的重组蛋白:全长anti-σ因子及截去羧基端部分序列的截短片段,包括Slr1055、Slr1055△(1~232)、Sll0688、Sll0688△(1~152)、Slr1546、Slr1546△(1~174)、Sll0857、Sll0857△(1~101)和大肠杆菌的S2P底物RseA(1~148).为下一步在体外重构S2P蛋白酶与底物的酶切体系、阐释蓝藻体内S2P介导的级联信号转导机制奠定了基础.
单 位：
College of Light Industry and Food Sciences,South China University of Technology,Guangzhou 510640,China
关键词：
site-2 protease%substrate%anti-σ factor%prokaryotic expression%purification
摘 要：
Nearly all cyanobacterial species contain genes encoding site-2-protease(S2P) homologs.Some S2P homologs have been reported to play essential roles in regulating stress response through intramembrane proteolysis of membranebound anti-sigma factors.However,the mechanism of action and substrates of S2P in Synechocystis sp.PCC6803,Sll0862,Slr0643,Sll0528 and Slr1821 remain unsolved.In this study,we focused on the four sigma factor-anti sigma factor pairs,SigEChlH(Slr1055),SigI(Sll0687)-Sll0688,SigG(Slr1545)-Slr1546 and SigH(Sll0856)-Sll0857.Using pET-30b(+) as vector,we constructed recombinant plasmids,optimized expression and successfully purified the full length and truncated fragment of putative substrates,including Slr1055,Slr1055Δ(1–232),Sll0688,Sll0688Δ(1–152),Slr1546,Slr1546Δ(1–174),Sll0857 and Sll0857Δ(1–101) as well as RseA(1–148),the S2P substrate from E.coli.This work may lay the foundation for constructing in vitro enzymatic digestion system to justify the relationship of various S2Ps with their putative substrates.
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