Description:
<jats:title>Abstract</jats:title><jats:p>The delivery of molecules into cells poses a critical problem that has to be solved for the development of diagnostic tools and therapeutic agents acting on intracellular targets. Cargos which by themselves cannot penetrate cellular membranes due to their biophysical properties can achieve cell membrane permeability by fusion to protein transduction domains (PTDs). Here, we engineered a universal delivery system based on PTD‐fused<jats:italic>Strep</jats:italic>‐Tactin, which we named Transtactin. Biochemical characterization of Transtactin variants bearing different PTDs indicated high thermal stabilities and robust secondary structures. Internalization studies demonstrated that Transtactins facilitated simple and safe transport of<jats:italic>Strep</jats:italic>‐tag II‐linked small molecules, peptides and multicomponent complexes, or biotinylated proteins into cultured human cells. Transtactin‐introduced cargos were functionally active, as shown for horseradish peroxidase serving as a model protein. Our results demonstrate that Transtactin provides a universal and efficient delivery system for<jats:italic>Strep</jats:italic>‐tag II‐fused cargos.</jats:p>