Intracellular delivery of glutathione S-transferase-fused proteins into mammalian cells by polyethylenimine-glutathione conjugates

Hitoshi Murata, Junichiro Futami, Midori Kitazoe, Takayuki Yonehara, Hidetaka Nakanishi, Megumi Kosaka, Hiroko Tada, Masakiyo Sakaguchi, Yasuyuki Yagi, Masaharu Seno, Nam Ho Huh, Hidenori Yamada

Research output: Contribution to journalArticle

22 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)447-455
Number of pages9
JournalJournal of Biochemistry
Volume144
Issue number4
DOIs
Publication statusPublished - Oct 2008

Fingerprint

Polyethyleneimine
Glutathione Transferase
Glutathione
Cells
Proteins
Signal transduction
Functional analysis
Molecular mass
Endocytosis
Green Fluorescent Proteins
Protein Kinases
Signal Transduction
Screening
Chemical activation
Derivatives
Imaging techniques

Keywords

  • Cationization
  • Glutathione
  • Glutathione S-transferase
  • Polyethylenimine
  • Protein transduction

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Intracellular delivery of glutathione S-transferase-fused proteins into mammalian cells by polyethylenimine-glutathione conjugates. / Murata, Hitoshi; Futami, Junichiro; Kitazoe, Midori; Yonehara, Takayuki; Nakanishi, Hidetaka; Kosaka, Megumi; Tada, Hiroko; Sakaguchi, Masakiyo; Yagi, Yasuyuki; Seno, Masaharu; Huh, Nam Ho; Yamada, Hidenori.

In: Journal of Biochemistry, Vol. 144, No. 4, 10.2008, p. 447-455.

Research output: Contribution to journalArticle

@article{6ff29e459a814e07bed3d0892fbe9200,
title = "Intracellular delivery of glutathione S-transferase-fused proteins into mammalian cells by polyethylenimine-glutathione conjugates",
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.",
keywords = "Cationization, Glutathione, Glutathione S-transferase, Polyethylenimine, Protein transduction",
author = "Hitoshi Murata and Junichiro Futami and Midori Kitazoe and Takayuki Yonehara and Hidetaka Nakanishi and Megumi Kosaka and Hiroko Tada and Masakiyo Sakaguchi and Yasuyuki Yagi and Masaharu Seno and Huh, {Nam Ho} and Hidenori Yamada",
year = "2008",
month = "10",
doi = "10.1093/jb/mvn087",
language = "English",
volume = "144",
pages = "447--455",
journal = "Journal of Biochemistry",
issn = "0021-924X",
publisher = "Oxford University Press",
number = "4",

}

TY - JOUR

T1 - Intracellular delivery of glutathione S-transferase-fused proteins into mammalian cells by polyethylenimine-glutathione conjugates

AU - Murata, Hitoshi

AU - Futami, Junichiro

AU - Kitazoe, Midori

AU - Yonehara, Takayuki

AU - Nakanishi, Hidetaka

AU - Kosaka, Megumi

AU - Tada, Hiroko

AU - Sakaguchi, Masakiyo

AU - Yagi, Yasuyuki

AU - Seno, Masaharu

AU - Huh, Nam Ho

AU - Yamada, Hidenori

PY - 2008/10

Y1 - 2008/10

N2 - 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.

AB - 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.

KW - Cationization

KW - Glutathione

KW - Glutathione S-transferase

KW - Polyethylenimine

KW - Protein transduction

UR - http://www.scopus.com/inward/record.url?scp=53549114484&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=53549114484&partnerID=8YFLogxK

U2 - 10.1093/jb/mvn087

DO - 10.1093/jb/mvn087

M3 - Article

VL - 144

SP - 447

EP - 455

JO - Journal of Biochemistry

JF - Journal of Biochemistry

SN - 0021-924X

IS - 4

ER -