Metabolic biotinylation of recombinant proteins in mammalian cells and in mice
Metabolic biotinylation of recombinant proteins in mammalian cells and in mice
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DOI：
10.1006/mthe.1999.0011
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发表时间：
2000-01-01
影响因子：
12.4
通讯作者：
Barry, MA
中科院分区：
文献类型：
--
作者：
Parrott, MB;Barry, MA
亲和素-生物素系统是生物医学中用于免疫定位、成像、核酸印迹和蛋白质标记的一项基本技术。虽然这项技术很强大，但它受到以下事实的限制：哺乳动物蛋白质必须在化学生物素化之前使用交联剂进行表达和纯化，交联剂可以将随机位置的蛋白质修饰到不同的水平，并可以使蛋白质功能失活。来绕过。在这种局限性下，我们证明了利用宿主的内源性生物素化酶在哺乳动物细胞和小鼠体内代谢生物素化标记蛋白的能力。使用单体亲和素可以很容易地从哺乳动物细胞中纯化内源性生物素化蛋白，并在非变性条件下仅使用生物素作为释放剂进行洗脱。这项技术应该能够从哺乳动物细胞以及转基因植物和动物中生产和纯化重组蛋白质和脆弱蛋白质复合体。此外，这项技术对于细胞靶向应用可能特别有用，在这种应用中，蛋白质或病毒基因治疗载体可以在基因定义的位置被生物素化，以便与其他与亲和素复合的靶向部分结合。
The avidin-biotin system is a fundamental technology in biomedicine for immunolocalization, imaging, nucleic acid blotting, and protein labeling. While this technology is robust, it is limited by the fact that mammalian proteins must be expressed and purified prior to chemical biotinylation using cross-linking agents which modify proteins at random locations to heterogeneous levels and can inactivate protein function. To circumvent. this limitation, we demonstrate the ability to metabolically biotinylate tagged proteins in mammalian cells and in mice using the endogenous biotinylation enzymes of the host. Endogenously biotinylated proteins were readily purified from mammalian cells using monomeric avidin and eluted under nondenaturing conditions using only biotin as the releasing agent. This technology should allow recombinant proteins and fragile protein complexes to be produced and purified from mammalian cells as well as from transgenic plants and animals. In addition, this technology may be particularly useful for cell-targeting applications in which proteins or viral gene therapy vectors can be biotinylated at genetically defined sites for combination with other targeting moieties complexed with avidin.