TAT-mediated delivery of human glutamate dehydrogenase into PC12 cells.
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.
Yoon
Hye-Young
HY
Department of Biochemistry, University of Ulsan College of Medicine, Seoul, South Korea.
Lee
Sung Ho
SH
Cho
Sung-Woo
SW
Lee
Jong Eun
JE
Yoon
Chang Sik
CS
Park
Jinseu
J
Kim
Tae Ue
TU
Choi
Soo Young
SY
D000595:Amino Acid Sequence; D000818:Animals; D015696:Gene Products, tat / Q000008:administration & dosage*; D005969:Glutamate Dehydrogenase / Q000008:administration & dosage / Q000378:metabolism*; D006678:HIV / Q000378:metabolism*; D006801:Humans; D016716:PC12 Cells; D051381:Rats; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D054322:tat Gene Products, Human Immunodeficiency Virus; D005969:Glutamate Dehydrogenase