(242g) Solubility Partner IF2 Domain I Enables High Yield Synthesis of Transducible Transcription Factor Fusion Proteins in E. Coli

Human induced pluripotent stem cells (iPSCs) have been successfully generated using both nucleic acid and transducible transcription factor fusion protein approaches. Protein-based iPSCs avoid genomic instability issues, but protein-based iPSC reprogramming efficiency is low and reprogramming, to date, has been performed using unpurified proteins in crude cell lysate. Thus, there is a need for more robust and efficient methods of non-viral nuclear reprogramming. The search for optimal reprogramming conditions will require large amounts of recombinant protein reagents. Producing soluble transducible transcription factors in these amounts is currently not feasible since a large fraction of the transducible transcription factor product that is expressed in bacterial hosts is insoluble.

We will discuss how we produced and purified milligram quantities of soluble OCT4, SOX2, NANOG, KLF4, and LIN28 transcription factors in vivo using a solubility fusion partner, IF2 Domain I (IF2D1). Though the transcription factor cargoes do not remain soluble after protease cleavage removal of IF2D1, the un-cleaved fusion transcription factors are able bind to their cognate DNA sequences. When we administer these un-cleaved fusion transcription factors to human foreskin fibroblast cultures, they provide partial stimulation of downstream gene targets.