带有穿膜肽重组PTD-KLF4的制备及活性鉴定

Preparation of Recombinant PTD-KLF4 with Penetrating Peptide and Activity Assay

KLF4是Kruppel样转录因子(kruppel-like factors,KLF)家族中的一员,是维持胚胎干细胞(embryonic stem cells,ESCs)全能性的重要转录因子。蛋白质转导结构域(protein transd uctiondomain,PTD)能够携带大分子进入细胞。为了获得具有穿膜功能的重组KLF4蛋白,利用原核表达载体PKYB表达KLF4与PTD的融合蛋白PTD-KLF4,用Ni柱纯化融合蛋白并作Westernblotting鉴定。利用异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记PTD-KLF4检测其穿越中国仓鼠卵巢(chinese hamster ovary,CHO)细胞细胞膜的能力;用荧光共振能量转移(fluorescence resonance energy transfer,FRET)检测重组融合蛋白PTD-KLF4结合目的DNA的活性。结果表明:重组PTD-KLF4可以成功进入细胞、定位细胞核内,穿膜效率为(22.29±2.1)%。重组融合蛋白PTD-KLF4引起细胞形态变化,并具有与目标DNA序列特异结合的能力。重组PTD-KLF4的制备为外源蛋白诱导多能干细胞(induced pluripotent stem cells,IPSCs)奠定基础。

KLF4 is one member of the kruppel-like factors（KLF） family,which is an important transcription factor to maintain the pluripotency of embryonic stem cells（ESCs）.Protein transduction domain（PTD） can carry macromolecules into cells.In order to obtain the recombinant KLF4 protein with penetrating membrane activity,the KLF4 gene was expressed as a fusion protein PTD-KLF4 combined with PTD using prokaryotic expression vector PKYB.The fusion protein PTD-KLF4 was purified by Ni affinity chromatography and identified by Western blotting analysis.The fluorescein isothiocyanate（FITC） labeled PTD-KLF4 was used to investigate the penetrating ability of the fusion protein into Chinese hamster ovary（CHO） cells.And the bioactivity of PTD-KLF4 binding the target DNA sequence was detected by fluorescence resonance energy transfer（FRET）.The results showed that the recombinant PTD-KLF4 successfully entered the cells and located in the nucleus with the transmembrane efficiency of（22.29±2.1）%.The recombinant PTD-KLF4 caused cell shape changes and had specific binding capacity with its target DNA sequence.The preparation of the recombinant PTD-KLF4 laid a good foundation for the induction of pluripotent stem cells（IPSCs） with exogenous protein.