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

23 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

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Fingerprint Dive into the research topics of 'Intracellular delivery of glutathione S-transferase-fused proteins into mammalian cells by polyethylenimine-glutathione conjugates'. Together they form a unique fingerprint.

  • Cite this