Journal of Biochemistry Advance Access originally published online on July 4, 2008
Journal of Biochemistry 2008 144(4):447-455; 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
1Department of Bioscience and Biotechnology, Faculty of Engineering, Graduate School of Natural Science and Technology; 2Engineering Innovation Center, Okayama University, Okayama 700-8530; 3Nippon Shokubai Co., Ltd., 5-8 Nishi Otabi-cho, Suita, Osaka 564-8512; 4Department of Cell Biology, Graduate School of Medicine and Dentistry, Okayama University, Okayama 700-8558; and 5Research Center for Biomedical Engineering, Okayama University, Okayama 700-8530, Japan
*To whom correspondence should be addressed. Fax: +81-86-251-8215, E-mail: yamadah{at}cc.okayama-u.ac.jp
Received March 28, 2008; Accepted June 27, 2008
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Abstract |
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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
Abbreviations: caMEK1, constitutively active mitogen-activated protein kinase kinase 1; EGF, epidermal growth factor; ERK1, mitogen-activated protein kinase 1; FBS, fetal bovine serum; GST, glutathione S-transferase; MBP, myelin basic protein; PEI, polyethylenimine; PI, propidium iodide; THF, tetrahydrofuran