Denatured and reversibly cationized p53 readily enters cells and simultaneously folds to the functional protein in the cells
Denatured and reversibly cationized p53 readily enters cells and simultaneously folds to the functional protein in the cells
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DOI：
10.1021/bi052642a
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发表时间：
2006-05-16
期刊：
影响因子：
2.9
通讯作者：
Yamada, Hidenori
中科院分区：
文献类型：
--
作者：
Murata, Hitoshi;Sakaguchi, Masakiyo;Yamada, Hidenori
阳离子化是将蛋白质内化到活细胞中的有力策略。另一方面，通过二硫键可逆阳离子化的变性蛋白质不仅可溶于水，而且能够在体外折叠成天然构象。当阳离子化的这些优势结合在一起时，我们开发了一种新的方法，将变性蛋白质递送到细胞中，同时让它折叠以在细胞内表达其功能。这种“细胞内折叠”方法增强了在大肠杆菌中表达的重组蛋白作为包涵体的效用;也就是说，包涵体中的重组蛋白通过可逆阳离子化溶解，所述可逆阳离子化通过半胱氨酸残基通过与氨基丙基甲硫代磺酸酯或吡啶基二硫代丙酰基聚乙烯亚胺的二硫键进行，然后与细胞一起孵育，而不进行体外折叠程序。作为模型蛋白，我们研究了人肿瘤抑制基因p53。用可逆阳离子化的p53处理p53缺失的Saos-2细胞显示，发生了作为细胞中p53活化的指示的所有事件，例如二硫键的还原，随后形成四聚体，定位到细胞核中，诱导p53靶基因，以及诱导细胞凋亡。这些结果表明，通过半胱氨酸残基的变性蛋白质的可逆阳离子化是将功能蛋白质递送到细胞中的替代方法。当天然折叠蛋白不易获得时，这种方法将非常有用。
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.