HIV-1 TAT-mediated protein transduction and subcellular localization using novel expression vectors.
Several novel prokaryotic and eukaryotic expression vectors were constructed for protein transduction and subcellular localization. These vectors employed an N-terminal stretch of 11 basic amino acid residues (47-57) from the human immunodeficiency virus type 1 (HIV-1) TAT protein transduction domain (PTD) for protein translocation and cellular localization. The vectors also contained a six-histidine (His(6)) tag at the N- or C-terminus for convenient purification and detection, and a multiple cloning site for easy insertion of foreign genes. Some heterologous genes including HSV-TK, Bcl-rambo, Smac/DIABLO and GFP were fused in-frame to TAT PTD and successfully overexpressed in Escherichia coli. The purified TAT-GFP fusion protein was able to transduce into the mammalian cells and was found to locate mainly in the cytosol when exogenously added to the cell culture medium. However, using a transfection system, mammalian-expressed TAT-GFP predominantly displayed a nuclear localization and nucleolar accumulation in mammalian cell lines. This discrepancy implies that the exact subcellular localization of transduced protein may depend on cell type, the nature of imported proteins and delivery approach. Taken together, our results demonstrate that a TAT PTD length of 11 amino acids was sufficient to confer protein internalization and its subsequent cellular localization. These novel properties allow these vectors to be useful for studying protein transduction and nuclear import.
Yang
Yonghui
Y
Department of Molecular and Cellular Biology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, PR China.
Ma
Jun
J
Song
Zhiyin
Z
Wu
Mian
M
D021581:Active Transport, Cell Nucleus; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015696:Gene Products, tat / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000737:chemistry; D049452:Green Fluorescent Proteins; D015497:HIV-1; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D010455:Peptides / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D014407:Tumor Cells, Cultured; D054322:tat Gene Products, Human Immunodeficiency Virus
D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; C412403:DIABLO protein, human; D015696:Gene Products, tat; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D024101:Mitochondrial Proteins; D019913:Nuclear Localization Signals; D010455:Peptides; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins