2018 Fiscal Year Final Research Report

Cell-penetrating proteins generated by modification with naturally occurring polycationic polymer

Research Project

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Project/Area Number	16H03284
Research Category	Grant-in-Aid for Scientific Research (B)
Allocation Type	Single-year Grants
Section	一般
Research Field	Biomolecular chemistry
Research Institution	Fukui Prefectural University
Principal Investigator	HAMANO Yoshimitsu 福井県立大学, 生物資源学部, 教授 (50372834)
Co-Investigator(Kenkyū-buntansha)	丸山千登勢福井県立大学, 生物資源学部, 講師 (20452120)
Research Collaborator	TAKEUCHI Yamato
Project Period (FY)	2016-04-01 – 2019-03-31
Keywords	ポリカチオン / ペプチド / 細胞膜透過性 / ポリリジン
Outline of Final Research Achievements	Generally, biomacromolecules including proteins and enzymes do not penetrate cell membrane due to their high molecular weights. However, it has been reported that, by modifications with polycationic peptides, the biomacromolecules such as protein show a cell-penetrating activity. In this study, we focused on ε-poly-L-lysine (ε-PL) as a naturally occurring polycationic peptide. To introduce a highly reactive group into the ε-PL molecule, an ε-PL producing bacterium was cultivated using the medium supplemented with OH-PEG4-azide. The resulting peptide, ε-PL-PEG4-azide was purified from the culture broth and used as the cell-penetrating peptide. A fluorescent protein used as a model enzyme was chemically modified with a DBCO group and then was attached to ε-PL-PEG4-azide via "click chemistry". Expectedly, we observed that the fluorescent protein modified with ε-PL was able to penetrate the cell membrane and localized in the cytosol in HeLa cells.
Free Research Field	応用微生物学
Academic Significance and Societal Importance of the Research Achievements	タンパク質・酵素をポリカチオン修飾する方法として、カチオン性ペプチドとの融合タンパク質、化学的合成されたポリカチオン性分子による修飾（化学的架橋体）が報告されている。しかし、前者の問題点は、カチオン性ペプチド導入に起因する異常タンパク質の形成であり、後者の問題点は、高い反応性官能基を持ったポリカチオン性分子の化学合成にかかる高いコストである。一方、我々の確立した方法は、微生物の力を利用してε-PLに高い反応性官能基を低コストに導入できることを特徴としており、得られたε-PL-PEG4-azideを用いてε-PLの高い生体膜透過性をタンパク質・酵素に付与できる画期的な技術と言える。