Denatured and reversibly cationized p53 readily enters cells and simultaneously folds to the functional protein in the cells

Hitoshi Murata, Masakiyo Sakaguchi, Junichiro Futami, Midori Kitazoe, Takashi Maeda, Hideki Doura, Megumi Kosaka, Hiroko Tada, Masaharu Seno, Nam Ho Huh, Hidenori Yamada

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Cationization is a powerful strategy for internalizing a protein into living cells. On the other hand, a reversibly cationized denatured protein through disulfide bonds is not only soluble in water but also able to fold to the native conformation in vitro. When these advantages in cationization were combined, we developed a novel method to deliver a denatured protein into cells and simultaneously let it fold to express its function within cells. This "in-cell folding" method enhances the utility of recombinant proteins expressed in Escherichia coli as inclusion bodies; that is, the recombinant proteins in inclusion bodies are solubilized by reversible cationization through cysteine residues by disulfide bonds with aminopropyl methanethiosulfonate or pyridyldithiopropionylpolyethylenimine and then incubated with cells without an in vitro folding procedure. As a model protein, we investigated human tumor-suppressor p53. Treatment of p53-null Saos-2 cells with reversibly cationized p53 revealed that all events examined as indications of the activation of p53 in cells, such as reduction of disulfide bonds followed by tetramer formation, localization into the nucleus, induction of p53 target genes, and induction of apoptosis of cells, occurred. These results suggest that reversible cationization of a denatured protein through cysteine residues is an alternative method for delivery of a functional protein into cells. This method would be very useful when a native folded protein is not readily available.

Original languageEnglish
Pages (from-to)6124-6132
Number of pages9
JournalBiochemistry
Volume45
Issue number19
DOIs
Publication statusPublished - May 16 2006

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Disulfides
Proteins
Recombinant Proteins
Cysteine
Inclusion Bodies
Escherichia coli
Conformations
p53 Genes
Tumors
Genes
Chemical activation
Cells
Apoptosis
Water
Neoplasms
In Vitro Techniques
methanethiosulfonate

ASJC Scopus subject areas

  • Biochemistry

Cite this

Denatured and reversibly cationized p53 readily enters cells and simultaneously folds to the functional protein in the cells. / Murata, Hitoshi; Sakaguchi, Masakiyo; Futami, Junichiro; Kitazoe, Midori; Maeda, Takashi; Doura, Hideki; Kosaka, Megumi; Tada, Hiroko; Seno, Masaharu; Huh, Nam Ho; Yamada, Hidenori.

In: Biochemistry, Vol. 45, No. 19, 16.05.2006, p. 6124-6132.

Research output: Contribution to journalArticle

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