类弹性蛋白标签在大肠杆菌周质空间中表达研究

The Expression of Elastin-like Polypeptide Tag in Periplasmic Space of Escherichia coli

旨在研究类弹性蛋白[I]_(40)(elastin-like polypeptide[I]_(40),ELP[I]_(40))在大肠杆菌周质空间的表达。构建表达载体pIG6LH/ELP[I]_(40)及pIG6LH/ELP[I]_(40)+Trx,分别将构建的表达载体转化入表达宿主菌E.coli BLR(DE3),IPTG诱导表达,采用可逆相变循环(Inverse transition cycling,ITC)技术纯化蛋白。测定ELP[I]_(40)及ELP[I]_(40)+Trx蛋白相变温度(T_t),检测ELP[I]_(40)、ELP[I]_(40)+Trx蛋白浓度及NaCl对相变温度的影响。结果显示,经3轮ITC纯化得到了ELP[I]_(40)、ELP[I]_(40)+Trx蛋白。分别测定ELP[I]_(40)和ELP[I]_(40)+Trx在10、25、50、75和100μmol/L浓度下的T_t,其T_t依次为31.5℃、29℃、27℃、26℃、25℃和31.8℃、29.5℃、27.5℃、26℃、25.5℃;测定了不同浓度的NaCl对T_t影响,在ELP[I]_(40)和ELP[I]_(40)+Trx终浓度为25μmol/L,NaCl浓度为0.25、0.5、0.75、1.00和1.25 mol/L时,分别使ELP[I]_(40)的T_t由29℃降至24.5℃、22℃、19℃、15℃和11.5℃,使ELP[I]_(40)+Trx的T_t由29.5℃降至25℃、23℃、20.2℃、15.5℃和11.8℃。在大肠杆菌周质空间表达的ELP[I]_(40)与胞内表达的具有相同的理化性质,ELP可作为在大肠杆菌周质空间表达的蛋白质分离纯化标签。

This work aims to investigate the expression of elastin-like polypeptide[I]40（ELP[I]40）in the periplasmic space of Escherichia coli. The expression vectors of pIG6LH/ELP[I]40 and pIG6LH/ELP[I]40＋Trx were constructed and transfected into E. coli BLR （DE3）and induced to express by IPTG. Recombinant ELP fusion protein were purified by inverse transition cycling（ITC）. Inverse temperature transition（Tt）of ELP[I]40 and ELP[I]40＋Trx protein were measured. The effects of the concentrations of ELP[I]40 and ELP [I]40＋Trx proteins and NaCl on phase-transition temperature were investigated. ELP[I]40 and ELP[I]40＋Trx protein were purified by 3 round ITC. When the protein concentration of ELP[I]40 and ELP[I]40＋Trx were 10 μmol/L,25 μmol/L,50 μmol/L,75 μmol/L,and 100 μmol/L,the Tt were 31.5℃,29℃,27℃,26℃,25℃ and 31.8℃,29.5℃,27.5℃,26℃,25.5℃ respectively. When the final concentrations of ELP[I]40 and ELP[I]40＋Trx were 25 mmol/L respectively and the NaCl concentration was 0.25 mol/L,0.5 mol/L,0.75 mol/L,1.00 mol/L,and 1.25 mol/L respectively,the Tt of ELP[I]40 decreased from 29℃ to 24.5℃,22℃,19℃,15℃,and 11.5℃respectively and the Tt of ELP[I]40＋Trx from 29.5℃ to 25℃,23℃,20.2℃,15.5℃,and 11.8℃ respectively. The ELP[I]40 in the periplasmic space of E. coli possessed the same physicochemical properties as the intracellular one,and the ELP could be used as an isolation and purification tag of expressed proteins in E. coli’s periplasmic space.