Transduction of Cu,Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into mammalian cells.
A human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene was fused with a gene fragment encoding the nine amino acid transactivator of transcription (Tat) protein transduction domain (RKKRRQRRR) of HIV-1 in a bacterial expression vector to produce a genetic in-frame Tat-SOD fusion protein. The expressed and purified Tat-SOD fusion protein in Escherichia coli can enter HeLa cells in a time- and dose-dependent manner when added exogenously in a culture media. Denatured Tat-SOD protein was transduced much more efficiently into cells than were native proteins. Once inside the cells, transduced Tat-SOD protein was enzymatically active and stable for 24 h. The cell viability of HeLa cells treated with paraquat, an intracellular superoxide anion generator, was increased by transduced Tat-SOD. These lines of results suggest that the transduction of Tat-SOD fusion protein may be one of the ways to replenish the Cu,Zn-SOD in the various disorders related to this antioxidant enzyme.
Kwon
H Y
HY
Department of Physiology, College of Medicine, Hallym University, Chunchon, South Korea.
Eum
W S
WS
Jang
H W
HW
Kang
J H
JH
Ryu
J
J
Ryong Lee
B
B
Jin
L H
LH
Park
J
J
Choi
S Y
SY
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015696:Gene Products, tat / Q000737:chemistry / Q000235:genetics*; D015497:HIV-1 / Q000737:chemistry*; D006801:Humans; D017354:Point Mutation; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins; D013482:Superoxide Dismutase / Q000235:genetics*; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D013482:Superoxide Dismutase