构建一种精确表达小分子蛋白的技术平台

Establishment of technical platform for exact micromolecular protein expression

目的:克服原核表达的小分子蛋白难以去除附加氨基酸的问题,为基因工程精确表达小分子蛋白提供一种简便有效的解决方案。方法:以73个氨基酸的小分子蛋白(黑色素瘤生长激活因子CXCL1的功能区)为例,用PCR方法扩增了基因,TA克隆到载体PETSUMO。测序验证后的重组质粒转化至表达菌BL21(DE3)中诱导表达,融合蛋白用基质辅助解吸电离飞行时间质谱仪进行串联质谱鉴定(MALDI-T0F-MS/MS),通过特异性的SUMO蛋白酶酶解载体SUMO蛋白,利用SUMO蛋白和其蛋白酶带6×His标签的性质,经镍螯合柱亲和层析将二者去除,以Western blotting证明目的小分子蛋白的分离。结果:①经测序,重组质粒无突变,TA克隆方向正确,重组载体构建成功。②MALDI-T0F-MS/MS证明融合蛋白由SUMO蛋白和CXCL1蛋白组成,除去SUMO蛋白后的表达终产物经Western blotting鉴定,与其相应的抗体有特异性结合,证明分离得到的小分子蛋白即为目的蛋白。结论:运用这一技术可将载体蛋白完全去除从而达到精确表达小分子蛋白的目的,在分子生物学的实验研究中有很好的应用前景。

Objective： To inhibit the extra amino acid in the expressed small proteins,a special prokaryotic expression technology was appliedl Methods： The CXCL1 domain gene amplified by polymerase chain reaction （PCR） was cloned into the prokaryotic expression vector PET SUMO. The recombinant expression plasmids were transformed into E. coli BL21 （DE3） and expressed. The expression products were analyzed by MALDI-TOF-MS/MS. The SUMO protein protease was used to removed the SUMO protein. The mat peptide of CXCL1 was testified by the western blotting. Results： （1） By sequencing the recombinant plasmids was well identified with correct base sequence and inserted direction. （2）The fusing protein and micromolecular protein was testified respectively by the MAL- DI-TOF-MS/MS and the western blotting. Conclusion： The micromolecular protein could be expressed exactly through this technical plat- form with corking applied foreground.