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Journal of Biochemistry Advance Access published online on July 4, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn087
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© 2008 The Japanese Biochemical Society

Intracellular Delivery of Glutathione S-Transferase-fused Proteins into Mammalian Cells by Polyethylenimine-Glutathione Conjugates

Hitoshi Murata1, Junichiro Futami1,4, Midori Kitazoe1, Takayuki Yonehara1, Hidetaka Nakanishi2, Megumi Kosaka1, Hiroko Tada1, Masakiyo Sakaguchi3, Yasuyuki Yagi2, Masaharu Seno1,5, Nam-ho Huh3 and Hidenori Yamada1,4,*

1Department of Bioscience and Biotechnology, Faculty of Engineering, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan, 2Nippon Shokubai Co., Ltd., 5-8 Nishi Otabi-cho, Suita, Osaka 564-8512, Japan, 3Department of Cell Biology, Graduate School of Medicine and Dentistry, Okayama University, Okayama 700-8558, Japan, 4Engineering Innovation Center, Okayama University, Okayama 700-853, Japan, and 5Research Center for Biomedical Engineering, Okayama University, Okayama 700-8530, Japan

*To whom correspondence should be addressed. Dr. Hidenori Yamada. Phone and FAX: +81-86-251-8215. E-mail: yamadah{at}cc.okayama-u.ac.jp.

Received March 28, 2008; Accepted June 27, 2008


  Abstract

The glutathione S-transferase (GST)-fused protein expression system has been extensively used to generate a large quantity of proteins and has served for functional analysis in vitro. In this study, we developed a novel approach for the efficient intracellular delivery of GST-fused proteins into living cells to expand their usefulness up to in vivo use. Since protein cationization techniques are powerful strategies for efficient intracellular uptake by adsorptive-mediated endocytosis, GST-fused proteins were cationized by forming a complex with a polycationic polyethylenimine (PEI)-glutathione conjugate. On screening of protein transduction, optimized PEI-glutathione conjugate for protein transduction was characterized by a partly oligomerized mixture of PEI with average molecular masses of 600 (PEI600) modified with multiple glutathiones, which could have sufficient avidity for GST. Furthermore, enhanced endosomal escape of transduced GST-fused proteins was observed when they were delivered with a glutathione-conjugated PEI600 derivative possessing a hydroxybutenyl moiety. These results were confirmed by both intracellular confocal imaging of GST-fused green fluorescent protein and activation of an endogenous growth signal transduction pathway by a GST-fused constitutively active mutant of a kinase protein. These PEI-glutathione conjugates seem to be convenient molecular tools for protein transduction of widely used GST-fused proteins.

Key Words: cationization, glutathione, glutathione S-transferase, polyethylenimine, protein transduction


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