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HIV‐1 TAT‐mediated protein transduction and subcellular localization using novel expression vectors 1
Author(s) -
Yang Yonghui,
Ma Jun,
Song Zhiyin,
Wu Mian
Publication year - 2002
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(02)03624-4
Subject(s) - subcellular localization , transduction (biophysics) , biology , fusion protein , green fluorescent protein , microbiology and biotechnology , nuclear localization sequence , transfection , protein subcellular localization prediction , n terminus , gene , peptide sequence , biochemistry , recombinant dna , cytoplasm
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.