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

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 beta 4 (T beta 4), was delivered to 293F Cells, which were permeabilized by a pore-forming toxin, streptolysin O, and resealed by Ca2+ after T beta 4 uptake. As a result, we successfully observed H-1-N-15 HSQC signals originating from the T beta 4, including those from the N-terminal acetylation, which had occurred inside the cell as a post-translational modification.