Cellular translocation of proteins by transportan
Cellular translocation of proteins by transportan
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DOI：
10.1096/fj.00-0780fje
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发表时间：
2001-04-27
期刊：
影响因子：
4.8
通讯作者：
Langel, Ü
中科院分区：
文献类型：
--
作者：
Pooga, M;Kut, C;Langel, Ü
分子量为30kda的蛋白质。（绿色荧光蛋白，GFP）至150 kDa（单克隆和多克隆抗体）偶联到细胞易位肽转运蛋白上。我们研究了由此产生的蛋白肽结构渗透到Bowes黑色素瘤、BRL和COS-7细胞中的能力。与转运蛋白偶联的重组GFP孵育0.5 ~ 3 h后，BRL和COS-7细胞的细胞膜内发现大部分GFP荧光，说明转运蛋白可以折叠态内化约30 kDa的共价连接蛋白。转运蛋白可以内化更大尺寸的共价偶联分子；也就是亲和素和抗体（高达150 kDa）。运输肽与其货物之间的共价键不是强制性的，因为链霉亲和素在15分钟内作为与生物素化运输蛋白的非共价复合物被转运到细胞中。在细胞内，传递的链霉亲和素首先主要位于靠近质膜的位置，随后分布到核周区域。大部分内化链霉亲和素局限于囊泡结构，但有相当一部分蛋白质分布在细胞质中。我们的数据表明，转运蛋白可以将蛋白质和其他亲水大分子传递到完整的哺乳动物细胞中，这一发现显示了作为科学和治疗目的的强大细胞传递载体的良好潜力。
Proteins with molecular masses ranging from 30 kDa. (green fluorescent protein, GFP) to 150 kDa (monoclonal and polyclonal antibodies) were coupled to the cellular translocating peptide transportan. We studied the ability of the resulting protein-peptide constructs to penetrate into Bowes melanoma, BRL, and COS-7 cells. After 0.5-3 h incubation with recombinant GFP coupled to transportan, most of the GFP fluorescence was found in intracellular membranes of BRL and COS-7 cells, which suggests that transportan could internalize covalently linked proteins of about 30 kDa in a folded state. Transportan could internalize covalently coupled molecules of even larger size; that is, avidin and antibodies, (up to 150 kDa). The covalent bond between the transport peptide and its cargo is not obligatory because streptavidin was translocated into the cells within 15 min as a noncovalent complex with biotinylated transportan. Inside the cells, the delivered streptavidin was first located mainly in close proximity to the plasma membrane and was later distributed to the perinuclear region. Most of the internalized streptavidin was confined to vesicular structures, but a significant fraction of the protein was distributed in the cytoplasm. Our data suggest that transportan can deliver proteins and other hydrophilic macromolecules into intact mammalian cells, and this finding demonstrates good potential as powerful cellular delivery vector for scientific and therapeutic purposes.