胸腺肽α1-Spl DnaX内含肽融合蛋白的切割动力学研究

Study on the Cleavage Dynamics of the Fusion Protein Thymosin α1-Spl DnaX Intein

目的:研究胸腺肽α1(Tα1)与Spl DnaX内含肽融合蛋白AS的体外切割动力学。方法:构建Tα1和SplDnaX内含肽的融合表达载体pET-AS,转化大肠杆菌BL21(DE3),经乳糖诱导获得可溶性表达的融合蛋白AS,用镍亲和层析纯化该蛋白;综合评价温度、β-巯基乙醇(β-ME)浓度和诱导切割时间对Spl DnaX内含肽介导融合蛋白AS自切割释放Tα1的影响。结果:在大肠杆菌中诱导表达了融合蛋白AS,经镍柱纯化获得该蛋白;随着温度升高和β-ME浓度增加,诱导切割时间延长,Spl DnaX内含肽介导的切割率逐渐增大;最终采用300 mmol/Lβ-ME切割24 h,融合蛋白的硫解切割率大于90%。结论:通过对Spl DnaX内含肽的诱导切割条件进行摸索,确定了最适宜的切割条件,为利用该方法制备Tα1和其他小分子多肽提供了技术基础。

Objective： To study the in vitro inducible cleavage dynamics of the fusion protein thymosin α1（Tα1）-Spl DnaX intein（AS）.Methods： The recombinant vector pET-AS expressing the fusion protein AS was constructed and transformed into E.coli BL21（DE3）.After being induced by lactose,corresponding soluble fusion protein AS was obtained and purified with Ni-sepharose affinity chromatographic column.The effects of the temperature,the concentration of β-mercaptoethanol（β-ME） and the cleavage time on the intein-mediated production of Tα1 were investigated comprehensively.Results： The fusion protein AS was successfully expressed and purified.As the temperature and the concentration of β-ME rising,and the cleavage time extending,the cleavage percentage mediated by Spl DnaX intein increased gradually.Induced by 300 mmol/L β-ME at 42℃ for 24 h,above 90% of AS was thiolyzed.Conclusion： The optimum cleavage condition mediated by Spl DnaX intein was determined,laying the foundation for production of Tα1 or other small peptides.