Abstracts

Protein transduction domains (PTDs) may be capable of transducing full-length fusion proteins across plasma cell membranes and the blood-brain-barrier (BBB). They are therefore considered a promising tool for the development of protein therapy aimed at the central nervous system. In this study we analysed the characteristics of TAT-PTD genetically fused to cystatin B (CSTB), the protein responsible for the Progressive Myoclonus Epilepsy of Unverricht-Lundborg disease (ULD)., A PCR product containing the [italic]EPM1[/italic] coding region was cloned into the pRSET vector containing the TAT-PTD sequence. A control protein was assembled by insertion of [italic]EPM1[/italic] cDNA into this vector, followed by removal of TAT-PTD.
TAT-PTD-CSTB and CSTB were expressed in bacterial cells, purified and transduced into COS-7 cells and human lymphoblasts. Whole cell lysates and cellular fractions were obtained for study by polyacrylamide gel electrophoresis and immunoblotting. Transduced (and control) cells were also used for analysis through immunofluorescence microscopy., Initial results pointed to an apparent time and concentration-dependent transduction of TAT-PTD-CSTB. However, careful investigation demonstrated that the nuclear localization of TAT-PTD-CSTB was an artifact of fixation and that the fusion protein was adhered to the plasma membrane., TAT-PTD-CSTB does not penetrate the cells. For this reason, even if TAT-PTD is likely able to cross the BBB, the fusion of this protein to CSTB cannot be used as a form of replacement of the intracytoplasmic protein missing in ULD. Importantly, we discuss precautions to avoid false-positive results when working with TAT-PTD for protein therapy of neurological diseases.,