TAT-mediated protein transduction into mammalian cells
TAT-mediated protein transduction into mammalian cells
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DOI：
10.1006/meth.2001.1186
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发表时间：
2001-07-01
期刊：
影响因子：
4.8
通讯作者：
Dowdy, SF
中科院分区：
文献类型：
--
作者：
Becker-Hapak, M;McAllister, SS;Dowdy, SF
哺乳动物细胞的操作已经通过表达载体的转染、显微注射或肽基模拟物的扩散来实现。虽然这些方法取得了一定的成功，但经典的操纵方法不容易受到监管，而且可能很费力。避免这些问题的一种方法是使用HIV TAT介导的蛋白转导。虽然这项技术最初是在1988年描述的，但在随后的10年里几乎没有改进的报道。在过去的几年中，已经采取了重要的步骤来推进该技术成为一种广泛适用的方法，该方法允许将全长蛋白质快速引入原代和转化细胞中。该技术需要合成融合蛋白，使用细菌表达载体将达特转导结构域连接到目的分子，然后在可溶或变性条件下纯化该融合蛋白。纯化的融合蛋白可以直接加入哺乳动物细胞培养物中或体内注射到小鼠中。蛋白质转导以浓度依赖性方式发生，在不到5分钟内达到最大细胞内浓度，转导群体中所有细胞之间的细胞内浓度几乎相等。全长达特融合蛋白已被用于解决许多生物学问题，涉及细胞周期进程，细胞凋亡和细胞结构。这里描述的是产生、分离和利用TAT融合蛋白来影响哺乳动物细胞的基本要求。一个详细的协议，生产和转导TAT-Cdc 42到原代细胞，以说明该技术。(C)北京：科学出版社.
Manipulation of mammalian cells has been achieved by the transfection of expression vectors, microinjection, or diffusion of peptidyl mimetics. While these approaches have been somewhat successful, the classic manipulation methods are not easily regulated and can be laborious. One approach to circumvent these problems is the use of HIV TAT-mediated protein transduction. Although this technology was originally described in 1988, few improvements were reported in the subsequent 10 years. In the last few years, significant steps have been taken to advance this technology into a broadly applicable method that allows for the rapid introduction of full-length proteins into primary and transformed cells. The technology requires the synthesis of a fusion protein, linking the TAT transduction domain to the molecule of interest using a bacterial expression vector, followed by the purification of this fusion protein under either soluble or denaturing conditions. The purified fusion protein can be directly added to mammalian cell culture or injected in vivo into mice. Protein transduction occurs in a concentration-dependent manner, achieving maximum intracellular concentrations in less than 5 min, with nearly equal intracellular concentrations between all cells in the transduced population. Full-length TAT fusion proteins have been used to address a number of biological questions, relating to cell cycle progression, apoptosis, and cellular architecture. Described here are the fundamental requirements for the creation, isolation, and utilization of TAT-fusion proteins to affect mammalian cells. A detailed protocol for production and transduction of TAT-Cdc42 into primary cells is given to illustrate the technique. (C) 2001 Academic Press.