细胞穿膜肽TAT以非融合形式递送蛋白质的能力探究

Exploring the ability of TAT,a cell-penetrating peptide,to deliver proteins in a non-fused form

穿膜肽TAT被证实可用于不同类型药物分子的递送,包括核酸、蛋白质和小分子药物等。TAT可以通过共价连接和非共价连接等形式递送分子。但是迄今TAT采用非共价连接形式递送蛋白质分子的报道较少,也未有研究比较过融合形式和非融合形式下TAT递送蛋白质能力的差异。为了探究非融合形式对于TAT递送蛋白质能力的影响,本文采用了荧光显微镜观察和流式细胞检测法在细胞水平上探究了TAT以非融合形式递送增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)进入人非小细胞肺癌细胞A549的能力,发现其可以穿膜递送EGFP,并且递送能力与TAT浓度呈现正相关。同时,本文还表达纯化了TAT与绿色荧光蛋白质EGFP的融合蛋白TAT-EGFP并考察了其穿膜能力。本文将两种形式的递送效果进行定量对比后发现:当TAT以融合蛋白形式递送EGFP时,其递送能力要明显优于TAT以非融合形式递送EGFP的能力。本文报道了TAT能够以非融合形式递送EGFP,尽管其递送效果有待进一步提高,但是该递送形式在操作便捷性上有着不可比拟的优势,是未来一种值得选择的递送方案。

Accumulating evidence has shown that the cell-penetrating peptide TAT can be applied to deliver different types of drug molecules,including nucleic acids,proteins and small molecule drugs.Usually TAT delivers cargoes on the basis of their covalent bonds or non-covalent interactions.However,there are few reports on the delivery of proteins by TAT in a non-covalent manner,and no quantitative comparisons have been made on the protein delivery ability of TAT in fusion and non-fusion manners.In order to explore the ability of TAT to deliver proteins in non-fusion manner,here we used fluorescence microscopy and flow cytometry to investigate the ability of TAT to deliver enhanced green fluorescent protein(EGFP)into non-small cell lung cancer cells A549 in a nonfusion manner.It was found that TAT could deliver EGFP into A549 cells,and its delivery ability was positively correlated with its concentration.In addition,the fusion protein TAT-EGFP was overexpressed and purified,and its permeability across cell membrane was also investigated.In this paper,based on quantitative comparison,we found that the delivery of EGFP by TAT in fusion manner is significantly efficient than that of TAT in non-fusion manner.This is the report that TAT can deliver EGFP in a non-fusion manner.Although its delivery efficiency remains to be improved as compared with the fusion manner,the non-fusion manner has shown incomparable advantages in ease of operation,suggesting that it is also a candidate for delivery strategy in the future.