Intracellular Delivery of Glutathione S-transferase-fused Proteins into Mammalian Cells by Polyethylenimine-Glutathione Conjugates
Autor: | Junichiro Futami, Midori Kitazoe, Hidenori Yamada, Hitoshi Murata, Masakiyo Sakaguchi, Yasuyuki Yagi, Megumi Kosaka, Nam Ho Huh, Takayuki Yonehara, Hiroko Tada, Masaharu Seno, Hidetaka Nakanishi |
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Rok vydání: | 2008 |
Předmět: |
BALB 3T3 Cells
Recombinant Fusion Proteins Green Fluorescent Proteins MAP Kinase Kinase 1 Biological Transport Active CHO Cells Biology Endocytosis Biochemistry Green fluorescent protein Mice chemistry.chemical_compound Transduction (genetics) Cricetulus Transduction Genetic Cricetinae Chlorocebus aethiops Animals Humans Polyethyleneimine Molecular Biology Glutathione Transferase Drug Carriers Polyethylenimine General Medicine Glutathione Molecular Weight Glutathione S-transferase chemistry COS Cells biology.protein Signal transduction Intracellular HeLa Cells |
Zdroj: | Journal of Biochemistry. 144:447-455 |
ISSN: | 0021-924X |
DOI: | 10.1093/jb/mvn087 |
Popis: | 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. |
Databáze: | OpenAIRE |
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