TAT-mediated delivery of human glutamate dehydrogenase into PC12 cells.

Abstract

Human glutamate dehydrogenase (GDH) gene was fused with a gene fragment encoding the nine amino acid (RKKRRQRRR) protein transduction domain of human immunodeficiency virus TAT protein in bacterial expression vector to produce genetic in-frame TAT–GDH fusion protein. The TAT–GDH protein can enter PC12 cells efficiently when added exogenously in culture media as determined by Western blot analysis and enzyme activities. Once inside the cells, the transduced denatured TAT–GDH protein showed a full activity of GDH indicating that the TAT–GDH fusion protein was correctly refolded after delivery into cells and the activities of GDH in the TAT–GDH fusion protein was not affected by the addition of the TAT sequence. TAT–GDH fusion protein and TAT itself showed no cytotoxicity in PC12 cells. Although the exact mechanism of transduction across a membrane remains unclear, the transduction activity of TAT–GDH into PC12 cells may suggest new possibilities for direct delivery of GDH into the patients with the GDH-deficient disorders.