PTD-mLumin-HMGB1 A原核蛋白的表达、纯化及初步鉴定

Expression,purification and primary identification of PTD-mLumin-HMGB1 A fusion protein

目的:构建含有蛋白转导结构域(protein transduction domain,PTD)和荧光蛋白mLumin的人HMGB1 A盒原核表达载体,表达并纯化PTD-mLumin-HMGB1 A融合蛋白。方法:利用基因重组技术构建pET28a-PTD-mLumin-HMGB1 A融合蛋白原核表达载体,并在大肠埃希菌Rosette(DE3)中表达融合蛋白,经Ni2+分离柱纯化PTD-mLumin-HMGB1 A融合蛋白,激光共聚焦显微镜观察其穿膜能力。结果:成功构建了PTD-mLumin-HMGB1 A原核表达载体,并表达和纯化了PTD-mLumin-HMGB1 A融合蛋白,该融合蛋白能高效地穿过细胞膜进入细胞内。结论:成功地表达和纯化了PTD-mLumin-HMGB1 A融合蛋白,此融合蛋白具有良好的穿膜能力,为更好地研究HMGB1 A的细胞内定位和体内定位提供了重要的依据。

Objective：To express and purify human HMGB1 A box fusion protein,PTD-mLumin-HMGB1 A,which contains a protein transduction domain and a newly isolated far-red fluorescent protein named mLumin,and investigate its ability to across the membrane.Methods：Expression vector of PTD-mLumin-HMGB1 A was constructed with inserting the HMGB1 A box into pET28a-PTD-mLumin vector.The fusion protein was expressed by E.coli Rossetta（DE3） and purified by Ni-column.Confocal microscopy was used to confirm the transmembrane ability of PTD-mLumin-HMGB1 A.Results：The vector of pET28a-PTD-mLumin-HMGB1 A was successfully constructed,and the fusion protein expressed and was purified efficiently.PTD-mLumin-HMGB1 A fusion protein transduced into EL-4 cell efficiently.Conclusion：The PTD-mLumin-HMGB1 A protein was successfully constructed,which laid the foundation for further investigation of biological function of human HMGB1 A box.