期刊
JOURNAL OF BIOSCIENCE AND BIOENGINEERING
卷 123, 期 4, 页码 419-424出版社
SOC BIOSCIENCE BIOENGINEERING JAPAN
DOI: 10.1016/j.jbiosc.2016.11.011
关键词
Protein transduction; Cell-permeable peptide; In-cell folding; Amphipathic peptide; Chemical modification
资金
- Ministry of Education, Culture, Sports, Science and Technology of Japan [23360370, 16H04580]
- Okayama Foundation for Science and Technology
- [24656506]
- Grants-in-Aid for Scientific Research [16H04580, 23360370] Funding Source: KAKEN
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 GAIA-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. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.
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