Intracellular delivery of glutathione S-transferase-fused proteins into mammalian cells by polyethylenimine-glutathione conjugates.
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.
Murata
Hitoshi
H
Department of Bioscience and Biotechnology, Faculty of Engineering, Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan.
Futami
Junichiro
J
Kitazoe
Midori
M
Yonehara
Takayuki
T
Nakanishi
Hidetaka
H
Kosaka
Megumi
M
Tada
Hiroko
H
Sakaguchi
Masakiyo
M
Yagi
Yasuyuki
Y
Seno
Masaharu
M
Huh
Nam-ho
NH
Yamada
Hidenori
H
D000818:Animals; D041702:BALB 3T3 Cells; D001693:Biological Transport, Active; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D004337:Drug Carriers; D004705:Endocytosis; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D011094:Polyethyleneimine / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic
D004337:Drug Carriers; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine; D005982:Glutathione Transferase; D048369:MAP Kinase Kinase 1; D005978:Glutathione