Development and application of in cell folding technology based on reversible cationization of denatured proteins.

基于变性蛋白可逆阳离子化的细胞内折叠技术的开发与应用

• 批准号: 17360399
• 负责人: YAMADA Hidenori
• 金额: $ 9.66万
• 依托单位: OKYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17360399/
• 关键词: Protein; chemical modification; protein transduction; folding; protein purfication; cell culture; protein engineering; biotechnology

This project aimed for the development of two methodologies; first is efficient solubilization of denatured proteins with reversible protein chemical cationization techniques, and second is intracellular delivery of reversibly cationized proteins followed by simultaneous folding to the active conformations, called in cell folding techniques. As a model protein, we investigated human tumor-suppressor p53. Bacterially expressed p53 protein as insoluble inclusion body was successfully solubilized by introduction of polyethylenimine 600 (PEI600, molecular weight: 600) via disulfide bond. The analysis of resulted PEI600-SS-p53 revealed that six cysteins were conjugated by PEI600, but remaining four cysteins failed in conjugation of PEI600, probably because of steric hindrance. This result indicated that reversible blocking of remaining free sulfidryl groups by a small molecule of aminopropyl methanethiosulfonate is important for preparation of chemically stable samples. The 'in cell folding' of p53 was successfully demonstrated by treatment of p53-null Saos-2 cells with reversibly cationized p53. PEI600-SS-p53 treated cells 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. Cationic charge dependent protein transduction efficiency was seen on the comparison between APS-SS-p53 (net charge: +6) and PEI600-SS-p53 (net charge: +81.6) because more cationic PEI600-SS-p53 showed more efficient induction of p53 targeted gene expression. This project also revealed that most of denatured proteins possessing cysteine residues can be solubilized by introduction of excess cationic charges. Furthermore, protein transduction and induction of their biological functions in living cells by using in cell folding techniques were confirmed by using at least 6 kinds of proteins.

该项目旨在开发两种方法;第一种是利用可逆蛋白质化学阳离子化技术有效溶解变性蛋白质，第二种是可逆阳离子化蛋白质的细胞内递送，然后同时折叠成活性构象，称为细胞折叠技术。作为模型蛋白，我们研究了人肿瘤抑制基因p53。通过二硫键引入聚乙烯亚胺600（PEI 600，分子量：600），成功地将细菌表达的p53蛋白作为不溶性包涵体溶解。对PEI 600-SS-p53的分析表明，6个半胱氨酸与PEI 600结合，但其余4个半胱氨酸未能与PEI 600结合，可能是由于空间位阻。该结果表明，通过小分子甲硫代磺酸氨丙酯可逆封闭剩余的游离硫基对于制备化学稳定的样品是重要的。通过用可逆阳离子化的p53处理p53缺失的Saos-2细胞成功地证明了p53的“细胞内折叠”。PEI 600-SS-p53处理的细胞显示，发生了作为细胞中p53活化的指示而检查的所有事件，例如二硫键还原，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。在APS-SS-p53（净电荷：+6）和PEI 600-SS-p53（净电荷：+81.6）之间的比较中观察到阳离子电荷依赖性蛋白转导效率，因为阳离子性更强的PEI 600-SS-p53显示更有效地诱导p53靶向基因表达。该项目还揭示了大多数具有半胱氨酸残基的变性蛋白质可以通过引入过量的阳离子电荷来增溶。此外，通过使用至少6种蛋白质，证实了通过使用细胞内折叠技术在活细胞中的蛋白质转导和诱导其生物学功能。

项目成果

Intracellular delivery of proteins into mammalian living cells by polyethylenimine-cationization

• DOI: 10.1263/jbb.99.95
• 发表时间: 2005-02-01
• 期刊: JOURNAL OF BIOSCIENCE AND BIOENGINEERING
• 影响因子: 2.8
• 作者: Futami, J;Kitazoe, M;Yamada, H
• 通讯作者: Yamada, H

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

• DOI: 10.1021/bi052642a
• 发表时间: 2006-05-16
• 期刊: BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Murata, Hitoshi;Sakaguchi, Masakiyo;Yamada, Hidenori
• 通讯作者: Yamada, Hidenori

Protein transduction assisted by polyethylenimine-cationized carrier proteins.

由聚乙烯亚胺阳离子化载体蛋白辅助的蛋白质转导。

• 发表时间: 2005
• 期刊: Journal of Biochemistry (Tokyo) 137
• 作者: Myagmar;B.E.;et al.;Sakaguchi et al.;Abarzua et al.;Takaishi et al.;Sakaguchi et al.;Kataoka et al.;Deguchi et al.;Futatmi et al.;Kitazoe et al.
• 通讯作者: Kitazoe et al.

Targeted disruption of transcriptional regulatory function of p53 by a novel efficient method for introducing a decoy oligonucleotide into nuclei

通过一种新的有效方法将诱饵寡核苷酸引入细胞核，有针对性地破坏 p53 的转录调节功能

• 发表时间: 2005
• 期刊: Nucleic Acids Research 33:e88
• 作者: Sakaguchi M;Nukui T;Sonegawa H;Murata H;Futami J;Yamada H;Huh NH
• 通讯作者: Huh NH