化学切割重组融合蛋白制备人胸腺肽β_4

Chemical Cleavage of Recombinant Fusion Protein to Prepare Human Thymosin β_4

目的:在大肠杆菌中表达人胸腺肽β4(Tβ4)的融合蛋白,通过CDAP介导的化学切割将融合部分切除,获得人Tβ4。方法:分别以质粒pET-Tβ4和pET-L12为模板,扩增Tβ4和核糖体亚基蛋白L12的基因片段,再以这2段基因为模板进行重叠PCR,并在连接处引入一个半胱氨酸(Cys)密码子,将得到的融合蛋白Tβ4-Cys-L12基因片段与pET-22b载体连接,构建表达质粒,将其与N-末端乙酰转移酶质粒共转化至大肠杆菌BL21(DE3)并共表达,获得N端乙酰化修饰的融合蛋白Tβ4-Cys-L12;利用CDAP氰基化Cys的巯基,于pH10.0条件下在Cys残基N端完成切割,分离纯化获得Tβ4。结果:质谱分析目的产物相对分子质量为4962.70,与天然Tβ4一致,表明获得了Tβ4。结论:CDAP介导的化学法可以有效切割融合蛋白获得Tβ4,建立了一套Tβ4的生物制备方法。

Objective： To obtain human thymosin β4 through chemical cleavage of recombinant fusion protein expressed in E.coli. Methods： Plasmid pET-Tβ4 and pET-L12 were employed as template to amplify the Tβ4 and L12 gene respectively, and then the fusion gene of Tβ4-Cys-L12 with a cystine codon in the linker region was amplified through overlap extension PCR. The fusion gene was then cloned into pET-22b to construct Tβ4-Cys-L12 expression vector which was transformed into E.coli BL21（DE3） together with a compatible plasmid harboring N-terminal acetyhransferase gene. The recombinant fusion protein completely acetylated by N-terminal acetyltransferase was cleaved under pil10.0 at the cystine residue. Results： Mass spectrum determined that the molecular weight of thymosin 134 was 4 962.70 Da, which was consistent with that of the natural thymosin 134. Conclusion： Chemical cleavage of recombinant fusion protein with the help of CDAP could effectively produce thymosin 134 which developed a new approach of producing human thymosin 134.