Tat-mediated protein transduction of human brain pyridoxal kinase into PC12 cells.
Pyridoxal kinase (PK) catalyses the phosphorylation of vitamin B6 to pyridoxal-5'-phosphate (PLP). A human brain PK 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-PK fusion protein. The expressed and purified Tat-PK fusion proteins transduced efficiently into PC12 cells in a time- and dose-dependent manner when added exogenously in culture media. Once inside the cells, the transduced Tat-PK proteins showed catalytic activity and are stable for 48 h. The intracellular concentration of PLP, which is known as a biologically active form of vitamin B6, was increased by pre-treatment of Tat-PK to the PC12 cells. Those results suggest that the transduction of Tat-PK fusion protein can be one of the ways to regulate the PLP level and to replenish this enzyme in the various neurological disorders related to vitamin B6.
Kim
Dae Won
DW
Department of Genetic Engineering and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702, South Korea.
Kim
Chung Kwon
CK
Choi
Soo Hyun
SH
Choi
Hee Soon
HS
Kim
So Young
SY
An
Jae Jin
JJ
Lee
Seung Ree
SR
Lee
Sun Hwa
SH
Kwon
Oh-Shin
OS
Kang
Tae-Cheon
TC
Won
Moo Ho
MH
Cho
Yong Joon
YJ
Cho
Sung-Woo
SW
Kang
Jung Hoon
JH
Kim
Tae Yoon
TY
Lee
Kil Soo
KS
Park
Jinseu
J
Eum
Won Sik
WS
Choi
Soo Young
SY
D000818:Animals; D001921:Brain / Q000201:enzymology*; D015696:Gene Products, tat / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D016716:PC12 Cells / Q000378:metabolism; D011731:Pyridoxal Kinase / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic; D014162:Transfection
D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D011731:Pyridoxal Kinase