1. Enhanced in-cell folding of reversibly cationized transcription factor using amphipathic peptide.
- Author
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Futami, Midori, Nakano, Tomoki, Yasunaga, Mayu, Makihara, Masahiro, Asama, Takashi, Hagihara, Yoshihisa, Nakajima, Yoshihiro, and Futami, Junichiro
- Subjects
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TRANSCRIPTION factors , *AMPHIPHILES , *PEPTIDES , *CELL membranes , *DISULFIDES - Abstract
The intracellular delivery of functionally active transcription factor proteins is emerging as a promising technique for artificial regulation of cellular functions. However, in addition to the cell membrane, which acts as a barrier to macromolecules, the aggregation-favored properties of structurally flexible transcription factor proteins limit the application of this method. In-cell folding technique can be used to overcome these issues. This technique solubilizes denatured protein by reversible alkyl-disulfide cationization ( S -cationization), and simultaneously endows efficient intracellular delivery and folding to the biologically active conformation in the reducing environment of the cytosol. Because cationized protein is internalized into cells by adsorption-mediated endocytosis, endosomal escape is crucial for this technique. In this study, we utilized a sensitive luciferase reporter gene assay to quantitatively evaluate in-cell folding of the artificial transcription factor GAL4-VP16. Although the cationic moiety of S -cationized protein was slightly affected, co-transduction of amphipathic peptide Endo-PORTER dramatically improved in-cell folding efficiency. Live cell imaging of fluorescent-labeled GAL4-VP16 revealed that some of the proteins diffused into the cytosol and nucleus through co-transduction with Endo-PORTER. Real-time monitoring of light output of luciferase revealed the kinetics of in-cell folding, supporting that endosomal-release assisted by Endo-PORTER was stimulated by endosome acidification. Because this method can transduce proteins uniformly and repeatedly into living cells, S -cationized transcription factor proteins are widely applicable for the artificial regulation of cellular functions. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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