title: Denatured and reversibly cationized p53 readily enters cells and simultaneously folds to the functional protein in the cells.
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.
authors: Department of Bioscience and Biotechnology, Faculty of Engineering, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan.
authors: Hitoshi
authors: H
authors: Murata
authors: Masakiyo
authors: M
authors: Sakaguchi
authors: Junichiro
authors: J
authors: Futami
authors: Midori
authors: M
authors: Kitazoe
authors: Takashi
authors: T
authors: Maeda
authors: Hideki
authors: H
authors: Doura
authors: Megumi
authors: M
authors: Kosaka
authors: Hiroko
authors: H
authors: Tada
authors: Masaharu
authors: M
authors: Seno
authors: Nam-ho
authors: NH
authors: Huh
authors: Hidenori
authors: H
authors: Yamada