TAT-mediated delivery of human glutamate dehydrogenase into PC12 cells

Neurochem Int. 2002 Jul;41(1):37-42. doi: 10.1016/s0197-0186(01)00138-3.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Gene Products, tat / administration & dosage*
  • Glutamate Dehydrogenase / administration & dosage
  • Glutamate Dehydrogenase / metabolism*
  • HIV / metabolism*
  • Humans
  • PC12 Cells
  • Rats
  • tat Gene Products, Human Immunodeficiency Virus

Substances

  • Gene Products, tat
  • tat Gene Products, Human Immunodeficiency Virus
  • Glutamate Dehydrogenase