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 language | English |
|---|---|
| Pages (from-to) | 6124-6132 |
| Number of pages | 9 |
| Journal | Biochemistry |
| Volume | 45 |
| Issue number | 19 |
| DOIs | |
| Publication status | Published - May 16 2006 |
Fingerprint
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 journal › Article
}
TY - JOUR
T1 - Denatured and reversibly cationized p53 readily enters cells and simultaneously folds to the functional protein in the cells
AU - Murata, Hitoshi
AU - Sakaguchi, Masakiyo
AU - Futami, Junichiro
AU - Kitazoe, Midori
AU - Maeda, Takashi
AU - Doura, Hideki
AU - Kosaka, Megumi
AU - Tada, Hiroko
AU - Seno, Masaharu
AU - Huh, Nam Ho
AU - Yamada, Hidenori
PY - 2006/5/16
Y1 - 2006/5/16
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=33646558305&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33646558305&partnerID=8YFLogxK
U2 - 10.1021/bi052642a
DO - 10.1021/bi052642a
M3 - Article
VL - 45
SP - 6124
EP - 6132
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 19
ER -