多聚精氨酸融合增强型绿色荧光蛋白制备方法及穿膜效果

为了方便细胞穿膜肽R9融合蛋白的可溶性表达及功能上的研究，构建了pSUMO (小分子泛素样修饰蛋白) -R9-EGFP (增强型绿色荧光蛋白) 原核表达载体。分别纯化EGFP及R9-EGFP蛋白后，作用于HepG2，细胞经流式细胞仪及激光共聚焦检测R9细胞穿膜肽的作用效果。实验结果显示在SUMO分子伴侣的作用下，R9-EGFP融合蛋白获得可溶性表达。经流式细胞仪检测，R9细胞穿膜肽可以快速有效的携带目的蛋白进入细胞内部且呈时间、剂量依赖性，大约1.5 h以后荧光强度进入平台期。共聚焦显微镜检测结果表明R9细胞穿膜肽可以有效携带EGFP进入HepG2细胞，并显示主要聚集在细胞浆内。同时体外经肝素抑制实验显示，肝素抑制R9-EGFP穿膜的效率达到50％。这些结果表明，可以利用pSUMO-R9/Ni-NTA表达纯化系统，快速、有效地表达出可溶性多聚精氨酸融合蛋白，同时R9细胞穿膜肽可以有效地携带目的蛋白进入细胞内，为进一步研究多聚精氨酸的穿膜机制提供了基础。

Preparation and penetrating effect of the polyarginine- enhanced green fluorescence protein fusion protein

The aim of the study is to establish a platform to deliver therapeutic proteins into target cells through a polyarginine-based cell penetrating peptide. To facilitate the expression of therapeutic proteins, a pSUMO (Small Ubiquitin-like Modifier)-R9-EGFP (enhanced green fluorescence protein) prokaryotic expression vector was constructed. After induction, the fusion protein SUMO-R9-EGFP was efficiently expressed. To validate the cell penetrating ability of the fusion protein, HepG2 cells were incubated with the purified R9-EGFP or EGFP protein as control, internalization of the fluorescent proteins was examined by either flow cytometry or confocal microscopy. The result obtained by flow cytometry showed that the R9-EGFP fusion protein could efficiently penetrate into the HepG2 cells in a dose and time-dependent manner. In contrast, the fluorescence was barely detected in the HepG2 cells incubated with EGFP control. The fluorescence intensity of the R9-EGFP treated cells reached plateau phase after 1.5 h. The result obtained by confocal microscopy shows that R9-EGFP efficiently entered into the HepG2 cells and was exclusively located in the cytoplasm, whereas, no fluorescence was detected in the cells incubated with the EGFP control. The heparin inhibition experiment showed that heparin could inhibit penetrating effect of the R9-EGFP protein by about 50%, suggesting that the penetrating ability of the fusion protein is heparin-dependent. In summary, the study has established a platform to deliver therapeutic proteins into target cells through a polyarginine-based penetrating peptide.