Tat-mediated protein transduction of human brain pyridoxine-5-P oxidase into PC12 cells.
Pyridoxine-5-P oxidase catalyses the terminal step in the biosynthesis of pyridoxal-5-P, the biologically active form of vitamin B6 which acts as an essential cofactor. Here, a human brain pyridoxine-5-P oxidase gene was fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) in a bacterial expression vector to produce a genetic in-frame Tat-pyridoxine-5-P oxidase fusion protein. Expressed and purified Tat-pyridoxine-5-P oxidase fusion protein transduced efficiently into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced Tat-pyridoxine-5-P oxidase protein showed catalytic activity and was stable for 48 h. Moreover, the formation of pyridoxal-5-P was increased by adding exogenous Tat-pyridoxine-5-P oxidase to media pre-treated with the vitamin B6 precursor pyridoxine. In addition, the intracellular concentration of pyridoxal-5-P was markedly increased when Tat-pyridoxal kinase was transduced together with Tat-pyridoxine-5-P oxidase into cells.These results suggest that the transduction of Tat-pyridoxine-5-P oxidase fusion protein presents a means of regulating the level of pyridoxal-5-P and of replenishing this enzyme in various neurological disorders related to vitamin B6.
Kim
So Young
SY
Department of Biomedical Sciences and Research, Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702, Korea.
An
Jae Jin
JJ
Kim
Dae Won
DW
Choi
Soo Hyun
SH
Lee
Sun Hwa
SH
Hwang
Seok-Il
SI
Kwon
Oh-Shin
OS
Kang
Tae-Cheon
TC
Won
Moo Ho
MH
Cho
Sung-Woo
SW
Park
Jinseu
J
Eum
Won Sik
WS
Lee
Kil Soo
KS
Choi
Soo Young
SY
D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000201:enzymology*; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016716:PC12 Cells; D011734:Pyridoxaminephosphate Oxidase / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D011734:Pyridoxaminephosphate Oxidase