胸腺素α1-胸腺五肽在大肠杆菌中的表达及表达条件优化

Expression and optimization of expression conditions of Tα1-TP5 fusion peptide in E.coli

目的探讨胸腺素α1-胸腺五肽(Tα1-TP5)在大肠杆菌中的表达及表达条件优化。方法将化学合成的Tα1-TP5基因与原核表达载体pGEX-4T-1融合并转化至E.coliBL21(DE3),异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。利用SDS-PAGE电泳和AlphaEase凝胶电泳图像分析系统研究培养基组成、诱导时机、诱导温度、诱导剂浓度及诱导时间等条件对融合蛋白表达量的影响。融合蛋白经GST琼脂糖珠亲和色谱纯化及重组肠激酶切割后,电喷雾质谱鉴定Tα1-TP5。结果选用TB培养基,在菌体对数生长中后期加入终浓度为0.05 mmol/L的IPTG,37℃诱导5 h,GST融合蛋白的表达量最高,占菌体总蛋白的35.8%,且主要以可溶形式表达。Tα1-TP5的分子质量与理论值相似。结论 Tα1-TP5成功在E.coli中表达,并确定了最佳表达条件。

Purpose To investigate the expression of Tα1-TP5 fusion peptide and optimization of the expression conditions.Methods The chemically synthesized Tα1-TP5 gene was fused with prokaryotic expression vector pGEX-4T-1 and transformed into E.coli BL21（DE3）,subsequently induced by IPTG.SDS-PAGE electrophoresis and AlphaEase gel electrophoresis image analysis system were used to analyze the influence of culture medium,induction starting time,induction temperature,inducer concentration and induction time on the expression level of target protein.GST fusion protein was purified by GST sepharose and cut by recombinant enterokinase.Tα1-TP5 was identified by ESI-MS.Results When using TB as the medium and adding final concentration of 0.05 mmol/L of IPTG into the middle and late logarithmic phase of bacteria to induce for 5 h at 37 ℃,the expression of GST fusion protein was the highest,accounting for 35.8% of the bacterial total protein,and mainly in a soluble form.Identified by ESI-MS,the molecular weight of Tα1-TP5 was identical with theoretical value.Conclusion Tα1-TP5 has been successfully expressed in E.coli and the expression conditions of the GST fusion protein were optimized.