title: Uniformly cationized protein efficiently reaches the cytosol of mammalian cells.
abstract: Protein cationization techniques are powerful protein transduction methods for mammalian cells. As we demonstrated previously, cationized proteins with limited conjugation to polyethylenimine have excellent ability to enter into cells by adsorption-mediated endocytosis [Futami, J., et al. (2005) J. Biosci. Bioeng. 99, 95-103]. In this study, we show that proteins with extensive and uniform cationization covering the protein surface reach the cytoplasm and nucleus more effectively than proteins with limited cationic polymers or proteins that are fused to cationic peptides. Although extensive modification of carboxylates results in loss of protein function, chicken avidin retains biotin-binding ability even after extensive amidation of carboxylates. Using this cationized avidin carrier system, the protein transduction ability of variously cationized avidins was investigated using biotinylated protein as a probe. The results revealed that cationized avidins bind rapidly to the cell surface followed by endocytotic uptake. Small amounts of uniformly cationized avidin showed direct penetration into the cytoplasm within a 15 min incubation. This penetration route seemed to be energy dependent and functioned under cellular physiological conditions. A biotinylated exogenous transcription factor protein that penetrated cells was demonstrated to induce target gene expression in living cells.
authors.affiliation: Department of Biomedical Engineering, Faculty of Engineering, Okayama University of Science, 1-1 Ridaicho, Okayama 700-0005, Japan.
authors.forename: Midori
authors.initials: M
authors.lastname: Futami
authors.forename: Yasuyoshi
authors.initials: Y
authors.lastname: Watanabe
authors.forename: Takashi
authors.initials: T
authors.lastname: Asama
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Murata
authors.forename: Hiroko
authors.initials: H
authors.lastname: Tada
authors.forename: Megumi
authors.initials: M
authors.lastname: Kosaka
authors.forename: Hidenori
authors.initials: H
authors.lastname: Yamada
authors.forename: Junichiro
authors.initials: J
authors.lastname: Futami
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001360:Avidin / Q000737:chemistry* / Q000378:metabolism*; D019921:Biotinylation; D002645:Chickens; D003600:Cytosol / Q000378:metabolism*; D004337:Drug Carriers / Q000737:chemistry* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport
chemical_list: D004337:Drug Carriers; D010455:Peptides; C485184:enhanced green fluorescent protein; D001360:Avidin; D049452:Green Fluorescent Proteins