Observation of NMR Signals from Proteins Introduced into Living Mammalian Cells by Reversible Membrane Permeabilization Using a Pore-Forming Toxin, Streptolysin O
Shinji Ogino†, Satoshi Kubo†, Ryo Umemoto†, Shuxian Huang†, Noritaka Nishida† and Ichio Shimada*†‡
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.