连接肽对融合蛋白ELP[I]_(30)-linker-eGFP相变的影响

The Impact of Linker Peptide on Phase Transition of Fusion Protein ELP[I]_(30)-linker-eGFP

研究G4S和Poly N连接肽对融合蛋白ELP[I]30-linker-eGFP相变的影响.将编码两种不同连接肽G4S和Poly N的绿色荧光蛋白(enhanced green fluorescent protein,eGFP)基因克隆到pET28-ELP[I]30表达载体中,在宿主菌E.coli BLR(DE3)中经IPTG诱导表达ELP[I]30-linker-eGFP,通过可逆相变循环(inverse transition cycling,ITC)及镍柱亲和层析纯化ELP[I]30-linker-eGFP蛋白.结果显示,成功构建、表达具有活性的两种连接肽的融合蛋白ELP[I]30-linker-eGFP,连接肽G4S使融合蛋白产生不可逆相变,而Poly N不影响融合蛋白可逆相变,该研究对类弹性蛋白标签的应用具有指导意义.

To investigate the effection of different linker peptides on the fusion ELP[Ⅰ]30-eGFP proteins＇ bioac- tivity and phase transition, the eGFP genes with encoding G4S and Poly N linker were inserted into pET28- ELP[Ⅰ]30expression vector respectively. The fusion proteins were expressed in E. coli BLR（DE3） induced by IPTG respectively, and were confirmed via SDS-PAGE and Western blot. The fusion proteins were purified via ITC （Inverse transition cycling） and Nickel column affinity chromatography respectively. The fusion pro- teins ELP[Ⅰ]30-1inker-eGFP were successfully expressed and purified with activity. The linker peptide G4S could cause fusion protein an irreversible phase change, and the linker peptide Poly N didn＇t affect the fusion pro- tein reversible phase transition, which laid the foundation for the application of ELP[Ⅰ]30.