In Vitro And In Vivo Studies On The Biological Effects Of Cell Penetrating Peptides TAT And R9

Cell-penetrating peptide,also known as protein transduction domain,is a kind of short peptide that can help some other biological macromolecules such as proteins and peptides go through the cell membrane.It can be directly used to guide therapeutic proteins and peptides with biological effects and activities maintained to target cells.Cell-penetrating peptides were first discovered in HIV-1.HIV-1 trans-activator protein(TAT)has a special ability to permeate the cell membrane and then enter cytoplasm.The core sequence of TAT includes 11 amino acids and the sequence is YGRKKRRQRRR.Once TAT have been discovered,many other peptides have been added to the CPP family,including the synthetic poly-arginine R9,which is a kind of cell penetrating peptide fused by nine arginine.Because cell penetrating peptides TAT and R9 have different amino acids sequence,so their transmembrane mechanism and efficacy are different.Therefore,when choosing cell penetrating peptide to achieve our goal,their properties will be considerate respectively for each cell.This study mainly compares the transmembrane efficiency,cytotoxicity and distribution of TAT and R9,respectively,which are fused with green fluorescent protein.After we cloned Pet-15b-TAT-EGFP plasmid,we transformed E.coli with TAT-EGFP and R9-EGFP plasmid,and TAT-EGFP and R9-EGFP fusion proteins were refined considering the temperature,time and inducer of protein purification.The fusion protein TAT-EGFP and R9-EGFP were prepared and purified by His-tag affinity purification,and their transmembrane activity were verified with U2 OS cell line.The transmembrane efficiency of TAT-EGFP and R9-EGFP in different cells were compared by different methods and their distribution in mice were examined.The results suggested that we constructed pHis-Tat-eGFP plasmid successfully.The best purification conditions for TAT-EGFP were 12 hours,28? and 0.8 mM inducer and the best purification conditions for R9-EGFP were 4 hours,28? and 0.8mM inducer.The bright green TAT-EGFP and R9-EGFP were prepared and purified by His-tag affinity purification,and we confirmed these transmembrane peptides can penetrate into the cells with their biological activities conserved.With inflorescencemicroscopic,cell counting and flow cytometry experiments,we found that R9-EGFP penetrating efficiency is higher than TAT-EGFP in MCF-7,A549 and Hela cell lines.After in vivo experiments in mice,we concluded that both penetrating peptides can permeate in organ cells and got through blood-brain barrier.In comparison,TAT-EGFP had better penetrating effects and had stronger florescence concentrated in organs but R9-EGFP's penetrating effects was weak.In conclusion,our research provides experiment evidences for the option of TAT and R9 penetrating peptides by comparing their cell penetrating efficacy with three different cancer cell lines and mice in vivo study.