Protein transduction assisted by polyethylenimine-cationized carrier proteins.
Previously, we have reported that cationized-proteins covalently modified with polyethylenimine (PEI) (direct PEI-cationization) efficiently enter cells and function in the cytosol [Futami et al. (2005) J. Biosci. Bioeng. 99, 95-103]. However, it may be more convenient if a protein could be delivered into cells just by mixing the protein with a PEI-cationized carrier protein having a specific affinity (indirect PEI-cationization). Thus, we prepared PEI-cationized avidin (PEI-avidin), streptavidin (PEI-streptavidin), and protein G (PEI-protein G), and examined whether they could deliver biotinylated proteins and antibodies into living cells. PEI-avidin (and/or PEI-streptavidin) carried biotinylated GFPs into various mammalian cells very efficiently. A GFP variant containing a nuclear localization signal was found to arrive even in the nucleus. The addition of a biotinylated RNase A derivative mixed with PEI-streptavidin to a culture medium of 3T3-SV-40 cells resulted in remarkable cell growth inhibition, suggesting that the biotinylated RNase A derivative entered cells and digested intracellular RNA molecules. Furthermore, the addition of a fluorescein-labeled anti-S100C (beta-actin binding protein) antibody mixed with PEI-protein G to human fibroblasts resulted in the appearance of a fluorescence image of actin-like filamentous structures in the cells. These results indicate that indirect PEI-cationization using non-covalent interaction is as effective as the direct PEI-cationization for the transduction of proteins into living cells and for expression of their functions in the cytosol. Thus, PEI-cationized proteins having a specific affinity for certain molecules such as PEI-streptavidin, PEI-avidin and PEI-protein G are concluded to be widely applicable protein transduction carrier molecules.
Kitazoe
Midori
M
Department of Bioscience and Biotechnology, Faculty of Engineering, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan.
Murata
Hitoshi
H
Futami
Junichiro
J
Maeda
Takashi
T
Sakaguchi
Masakiyo
M
Miyazaki
Masahiro
M
Kosaka
Megumi
M
Tada
Hiroko
H
Seno
Masaharu
M
Huh
Nam-ho
NH
Namba
Masayoshi
M
Nishikawa
Mitsuo
M
Maeda
Yoshitake
Y
Yamada
Hidenori
H
D000818:Animals; D001360:Avidin; D041702:BALB 3T3 Cells; D001709:Biotechnology / Q000379:methods*; D019921:Biotinylation; D002352:Carrier Proteins / Q000378:metabolism; D002412:Cations / Q000737:chemistry / Q000378:metabolism; D016923:Cell Death; D005347:Fibroblasts; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins; D011094:Polyethyleneimine / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D012259:Ribonuclease, Pancreatic; D019809:Streptavidin
D002352:Carrier Proteins; D002412:Cations; C029152:G-substrate; D009419:Nerve Tissue Proteins; D001360:Avidin; D011094:Polyethyleneimine; D019809:Streptavidin; D012259:Ribonuclease, Pancreatic