【摘要】 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。更多还原

【Abstract】 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（T_t） 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 3round 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,and100 μmol/L,the T_t 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,0.75mol/L,1.00 mol/L,and 1.25 mol/L respectively,the T_t of ELP[I]₄₀ decreased from 29℃ to 24.5℃,22℃,19℃,15℃,and 11.5℃respectively and the T_t 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.更多还原