Yang Li-ping, Liu Zhi-feng, Li Yong-ming, Li Zhi-jie, Jiang Yong
Department of Pathophysiology and Guangdong Provincial Key Laboratory of Functional Proteomics, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2006 May;26(5):553-7.
To construct a p38 MAPK protein delivery system based on TAT protein and study its functions in eukaryotic cells.
Recombinant vectors pHis-TAT-p38 and pHis-TAT-p38(AF) were constructed, and two recombinant proteins, His-TAT-p38 and His-TAT-p38(AF), were expressed and purified in E. coli. The two fusion proteins were then incubated with ECV304 cells, respectively. The phosphorylation of ATF2 was detected to assay the effect of His-TAT-p38 on endogeneious p38 activity after the cells were stimulated by sorbitol.
The results of restriction enzyme digestion and DNA sequencing showed that the two recombinant vectors were correctly constructed. The recombinant proteins of His-TAT-p38 and His-TAT-p38(AF) were isolated and purified by SDS-PAGE, and Western blotting suggested that His-TAT-p38 and its mutant with dual phosphorylation sites could enter the cells efficiently in a time- and concentration-dependent manner. His-TAT-p38 was found capable of increasing the activity of endogenous p38 in ECV304 cells, but His-TAT-p38(AF) inhibited the phosphorylation of ATF2 so as to block the transduction of p38 signal pathway when the cells were stimulated with sorbitol.
p38 MAPK protein delivery system based on TAT protein has been constructed successfully. It is confirmed that TAT can transfer the proteins into the cells in a time- and dose-depended manner. TAT-p38 and its dominant negative form possess high biological activity after transduction into ECV304 cells by TAT protein delivery system, and the former can increase the activity of endogenous ATF2, but the latter inhibits the transduction of endogeneious p38 signal pathway in ECV304 cells with high osmotic stress.
构建基于TAT蛋白的p38丝裂原活化蛋白激酶（p38 MAPK）蛋白递送系统，并研究其在真核细胞中的功能。
构建重组载体pHis-TAT-p38和pHis-TAT-p38(AF)，并在大肠杆菌中表达和纯化两种重组蛋白His-TAT-p38和His-TAT-p38(AF)。然后将这两种融合蛋白分别与ECV304细胞孵育。在用山梨醇刺激细胞后，检测ATF2的磷酸化水平，以分析His-TAT-p38对内源性p38活性的影响。
限制性内切酶消化和DNA测序结果表明两种重组载体构建正确。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离并纯化了His-TAT-p38和His-TAT-p38(AF)重组蛋白，蛋白质免疫印迹法（Western blotting）显示His-TAT-p38及其双磷酸化位点突变体能够以时间和浓度依赖性方式高效进入细胞。发现His-TAT-p38能够增加ECV304细胞内源性p38的活性，但在用山梨醇刺激细胞时，His-TAT-p38(AF)抑制了ATF2的磷酸化，从而阻断了p38信号通路的转导。
成功构建了基于TAT蛋白的p38 MAPK蛋白递送系统。证实TAT能够以时间和剂量依赖性方式将蛋白转运到细胞中。通过TAT蛋白递送系统转导至ECV304细胞后，TAT-p38及其显性负性形式具有较高的生物学活性，前者可增加内源性ATF2的活性，而后者在高渗应激下抑制ECV304细胞内源性p38信号通路的转导。
