Transmembrane delivery and oxygen-dependent degradation mediated by trans-activator-oxygen-dependent degradation fused motif

OBJECTIVE To evaluate transmembrane activity of trans-activator(TAT) protein transduction domain and effect of oxygen-dependent degradation(ODD) domain under different oxygen tension microenvironments in vitro and in vivo.METHODS An enhanced green fluorescent protein(EGFP) fusion protein conjugate with TAT and ODD was constructed,expressed and purified via a series of molecular biological procedures.EGFP and TAT-EGFP were also prepared following similar manipulation.To assay the cell-permeability and hypoxia-targeting stability in vitro,various tumor cell lines including H1299,A549 and MDA-MB-231 were cultured with different EGFP fusion proteins under 20% and 1% oxygen tension conditions,respectively.The treated cells were fixed and then directly observed by fluorescence microscope.To evaluate the oxygen-dependent stability and the profile of distribution of TAT-ODD-EGFP in vivo,male BALB/c mice were iv given EGFP,TAT-EGFP and TAT-ODD-EGFP 10 mg·kg-1.Animals were executed to harvest organs,including the heart,liver,spleen,lung,kidney,intestine,and brain at 1,6 and 12 h after treatment.The frozen sections were prepared for assay by fluorescence microscope.RESULTSTAT-EGFP could be effectively delivered into and stabilized in the different cell lines in vitro and all tissues of mice were observed in vivo.Like TAT-EGFP,TAT-ODD-EGFP could be transformed into cells by TAT,but its stability was oxygen-dependent because of its degradation property from ODD under different oxygen tensions.CONCLUSION TAT can deliver its fusion proteins into cells and animal tissues effectively.TAT fusion protein conjugates with ODD was stabilized in hypoxic cells and tissues,but it was degrades quickly in normoxia because of ODD′s function.TAT-ODD domain can be used in the targeted transduction of anti-tumor proteins into hypoxic tumor tissues.