© 2005 The Japanese Biochemical Society
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Protein Transduction Assisted by Polyethylenimine-Cationized Carrier Proteins
1 Department of Bioscience and Biotechnology, Faculty of Engineering, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530; 2 Nippon Shokubai Co., Ltd.; 3 Department of Cell Biology, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558; 4 Research Center for Biomedical Engineering, Okayama University, Okayama 700-8530; 5 Niimi College, Niimi, Okayama 718-8585; and 6 KIRIN Brewery Co., LTD, Pharmaceutical Division, Pharmaceutical Research Laboratories, Takasaki, Gunma 370-1295
* To whom correspondence should be addressed. Tel: +81-86-251-8215, Fax: +81-86-251-8265, E-mail: yamadah{at}cc.okayama-u.ac.jp
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.
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  H. Murata, J. Futami, M. Kitazoe, T. Yonehara, H. Nakanishi, M. Kosaka, H. Tada, M. Sakaguchi, Y. Yagi, M. Seno, et al. Intracellular Delivery of Glutathione S-transferase-fused Proteins into Mammalian Cells by Polyethylenimine-Glutathione Conjugates J. Biochem., October 1, 2008; 144(4): 447 - 455. [Abstract] [Full Text] [PDF]
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