A TAT-streptavidin fusion protein directs uptake of biotinylated cargo into mammalian cells
A TAT-streptavidin fusion protein directs uptake of biotinylated cargo into mammalian cells
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DOI：
10.1093/protein/gzi014
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发表时间：
2005-03-01
影响因子：
2.4
通讯作者：
Stayton, PS
中科院分区：
文献类型：
--
作者：
Albarran, B;To, R;Stayton, PS
HIV-1达特肽已广泛用于指导各种货物（包括DNA、脂质体和大分子）的细胞内递送。对于蛋白质递送，已经描述了将达特编码序列连接至感兴趣的蛋白质编码序列的多种TAT融合蛋白。链霉亲和素代表了一种潜在有用的TAT融合蛋白，因为它可用于递送广泛的生物素化货物。在这里，我们已经表征了TAT-链霉亲和素（TAT-SA）融合蛋白，其保留了结合生物素化货物的能力，同时指导其有效的细胞摄取。荧光激活细胞分选（FACS）分析和共聚焦显微镜表征表明，TAT-SA被Jurkat T细胞和NIH 3 T3细胞单独内化，以及当与藻红蛋白复合时内化，而天然链霉亲和素则不内化。此外，生物素化的碱性磷酸酶被成功内化，并在与TAT-SA复合并与Jurkat T细胞孵育时保留其活性。然而，共聚焦显微镜表明，内化的TAT-SA和TAT-SA复合物主要是在囊泡隔室，而不是在细胞质隔室中自由扩散。为了实现细胞质递送，将内体释放聚合物聚（丙基丙烯酸）（PPAA）生物素化并与TAT-SA复合。荧光标记的TAT-SA与PPAA复合物的内体释放和细胞质递送通过荧光蛋白在细胞质中的弥散分布来显示。总之，这些结果表明，TAT-SA可用于将大的生物素化的货物引导细胞内递送至细胞内区室，并且生物素化的PPAA可在需要时引导细胞质递送。
The HIV-1 TAT peptide has been used extensively for directing the intracellular delivery of an assortment of cargo, including DNA, liposomes and macromolecules. For protein delivery, a variety of TAT-fusion proteins have been described which link the TAT coding sequence to the protein coding sequence of interest. Streptavidin represents a potentially useful TAT-fusion protein because it could be used to deliver a wide array of biotinylated cargo. Here we have characterized a TAT-streptavidin (TAT-SA) fusion protein, which retains the ability to bind biotinylated cargo while directing their efficient cellular uptake. Fluorescence activated cell sorting (FACS) analysis and confocal microscopy characterization showed that TAT-SA is internalized by Jurkat T-cells and NIH 3T3 cells alone and when complexed to phycoerythrin, whereas the native streptavidin is not. Additionally, biotinylated alkaline phosphatase is successfully internalized and retains its activity when complexed to TAT-SA and incubated with Jurkat T-cells. Confocal microscopy suggested, however, that internalized TAT-SA and TAT-SA complexes were largely compartmentalized in vesicular compartments, rather than freely diffusing in the cytoplasmic compartment. To effect cytoplasmic delivery, the endosomal releasing polymer, poly(propylacrylic acid) (PPAA), was biotinylated and complexed to TAT-SA. Endosomal release and cytoplasmic delivery of fluorescently labeled TAT-SA complexes with PPAA was shown by the diffuse distribution of fluorescent protein in the cytoplasm. Taken together, these results demonstrate that TAT-SA can be used to direct intracellular delivery of large biotinylated cargo to intracellular compartments and that biotinylated PPAA can direct cytoplasmic delivery where desired.