Intracellular delivery of a Tat-eGFP fusion protein into muscle cells

The Tt protein from HIV-1, when fused with heterologous proteins or peptide s, can traverse biological membranes in a process call ed "protein transduc tion," delivering its cargo into cells. A Tat-eGFP fusion protein was purif ied from bacteria to study the transduction kinetics of Tat fusion proteins into cultured myoblasts and in the muscle tissue. Correctly folded Tat-eGF P reaches a maximum intracellular level in nearly 30 min, while its endogen ous fluorescence is first detected only after 14 h. The nuclear localizatio n signal from the basic domain of Tat was not sufficient to confer nuclear localization to Tat-eGFP, suggesting that the nuclear import pathway used b y the exogenously added Tat-eGFP might be sensitive to the folding state of eGFP. In mice, the direct delivery to the muscle tissue using subcutaneous injections or the intra-arterial pathway led to few positive fibers in the muscle periphery or surrounding the blood vessels. Muscles injected with T at-eGFP showed intense labeling of the extracellular matrix (ECM), suggesti ng that, although Tat fusion proteins can transduce muscle fibers, their bi nding by components of the ECM surrounding myofibers could interfere with t he intracellular transduction process.