Observation of NMR Signals from Proteins Introduced into Living Mammalian Cells by Reversible Membrane Permeabilization Using a Pore-Forming Toxin, Streptolysin O

• Published in: Journal of the American Chemical Society Vol. 131; no. 31; pp. 10834 - 10835
• Main Authors: Ogino, Shinji, Kubo, Satoshi, Umemoto, Ryo, Huang, Shuxian, Nishida, Noritaka, Shimada, Ichio
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 12.08.2009
• Subjects: Bacterial Proteins - pharmacology; Calcium; Cell Line; Cell Membrane Permeability - drug effects; Humans; Methods; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular - methods; Proteins - chemistry; Proteins - pharmacokinetics; Streptolysins - pharmacology; Thymosin - chemistry; Thymosin - pharmacokinetics
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja904407w

We have developed a new in-cell NMR method that is applicable to any type of cell and does not require target protein modification or specialized equipment. The stable-isotope-labeled target protein, thymosin β4 (Tβ4), was delivered to 293F cells, which were permeabilized by a pore-forming toxin, streptolysin O, and resealed by Ca2+ after Tβ4 uptake. As a result, we successfully observed 1H−15N HSQC signals originating from the Tβ4, including those from the N-terminal acetylation, which had occurred inside the cell as a post-translational modification.