Yang Yonghui, Ma Jun, Song Zhiyin, Wu Mian
Department of Molecular and Cellular Biology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, PR China.
FEBS Lett. 2002 Dec 4;532(1-2):36-44. doi: 10.1016/s0014-5793(02)03624-4.
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.
构建了几种新型的原核和真核表达载体用于蛋白质转导和亚细胞定位。这些载体利用了来自人类免疫缺陷病毒1型（HIV-1）TAT蛋白转导结构域（PTD）的N端11个碱性氨基酸残基（47-57）片段来进行蛋白质转运和细胞定位。载体在N端或C端还含有一个六组氨酸（His(6)）标签，便于纯化和检测，以及一个多克隆位点，便于插入外源基因。一些异源基因，包括单纯疱疹病毒胸苷激酶（HSV-TK）、Bcl-rambo、Smac/DIABLO和绿色荧光蛋白（GFP），与TAT PTD进行读码框融合，并在大肠杆菌中成功过表达。纯化的TAT-GFP融合蛋白能够转导进入哺乳动物细胞，当外源添加到细胞培养基中时，发现其主要定位于细胞质溶胶中。然而，使用转染系统时，哺乳动物表达的TAT-GFP在哺乳动物细胞系中主要表现为核定位和核仁积累。这种差异表明，转导蛋白的确切亚细胞定位可能取决于细胞类型、导入蛋白的性质和递送方式。综上所述，我们的结果表明，11个氨基酸长度的TAT PTD足以赋予蛋白质内化及其随后的细胞定位。这些新特性使得这些载体可用于研究蛋白质转导和核输入。
