Intracellular reactivation of transcription factors fused with protein transduction domain

Induction of a desired cell type by defined transcription factors (TFs) using iPS technology can be used for cell replacement therapy. However, to overcome problems such as tumor formation, genomic insertional mutagenesis by viral transduction in the induction process needs to be avoided using alternative approaches. One approach could be the direct delivery of TF protein by a protein transduction system, whereby a protein transduction domain (PTD) is fused to facilitate the penetration of cell membrane. However, fusion proteins, including TFs, are reported to be biologically less active through the interference of PTD with proper protein folding. Here, we report a proof-of-concept study in which TF proteins fused with PTDs could be reactivated by removal of PTDs from cells. We demonstrated that Sox2 and Oct3/4 proteins fused with PTD were less active in mouse embryonic stem cells. Removal of PTD by a site-specific protease, derived from tobacco etch virus (TEV), substantially restored the functionality of these proteins, proved by enhanced rescue ability for differentiation induced by endogenous Sox2 and Oct3/ 4 repression. These results suggest that, by removing a PTD inside the cells, directly delivered TF proteins may exert substantially enhanced function than presently considered.