Penetrating Effect Of Arg9-EGFP Green Fluorescent Protein

The penetration of drug moleculers penetrate into the cells is a very important step in drug delivery system. Because mammalian cells contain the membrane,and many bioactive macromolecular drugs, such as proteins and nucleic acids can not directly get into cells, resulting in limitations in practice for the usage of drugs and difficulty to reach to achieve therapeutic effects.Cell penetrate peptide(CPPs) can carry a variety of active molecules efficiently into the cell. Over the past 20 years, different types of CPPs are reported. Based on their structure and biological cheminstry, the CPPs are found to poccess the following common characteristics:(1) Containing a different length of positively charged peptides, in which are rich with arginine, lysine or other basic amimo acid;(2) Presenting an alpha helix in their second structure. Among them, the CPP rich with arginine has been broadly studied. It is found that the CPP containing a single poly arginine can mediate the penetration of protein.To study penetration effects of Arg9 peptide, we constructed p ET-15b-Arg9-EGFP(enhanced green fluorescent protein) prokaryotic expression vector. With the IPTGinduced DE3 E.coli prokaryotic expression system and the His-tag purification system, we purified Arg9-EGFP fusion protein. The penetration effects of this Arg9-EGFP protein were tested in MCF-7 cells. Experimental results confirmed that the Arg9-EGFP fusion protein was produced successfully and it penetrated into cells efficiently in a dose-dependent manner. Fluorescence detection results shown that the Arg9-EGFP protein located mainly in the cytoplasm of MCF-7 cells. These results indicated that the Arg9 peptide was able to mediate target proteins to penetrate the cell membrane effectively and maintain the activities of the proteins. It provided a potential tool to mediate penetration of certain proteins resulting in therapeutical effects in cells.