Parrott M B, Barry M A
Department of Immunology, Baylor College of Medicine, Houston, Texas, USA.
Biochem Biophys Res Commun. 2001 Mar 9;281(4):993-1000. doi: 10.1006/bbrc.2001.4437.
Due to its strength and specificity, the interaction between avidin and biotin has been used in a variety of medical and scientific applications ranging from drug targeting to immunohistochemistry. To maximize the application of this technology in mammalian systems, we recently demonstrated the ability to metabolically biotinylate tagged proteins in mammalian cells using the endogenous biotin ligase enzymes of the mammalian cell. This technology allows site-specific biotinylation without any exogenous reagents and eliminates possible inactivation of the protein of interest by nonspecific biotinylation. Here, we report further expansion of the mammalian metabolic biotinylation technology to enable biotinylation of proteins secreted from mammalian cells and expressed on their cell surface by cosecretion with BirA, the biotin ligase of E. coli. This technique can be used to biotinylate secreted proteins for purification or targeting and also for biotinylating the surfaces of mammalian cells to facilitate their labeling and purification from other nontagged cells.
由于抗生物素蛋白与生物素之间相互作用的强度和特异性，其已被应用于从药物靶向到免疫组织化学等各种医学和科学应用中。为了在哺乳动物系统中最大限度地应用这项技术，我们最近证明了利用哺乳动物细胞的内源性生物素连接酶在哺乳动物细胞中对标记蛋白进行代谢性生物素化的能力。这项技术允许进行位点特异性生物素化，无需任何外源试剂，并消除了因非特异性生物素化导致目标蛋白可能失活的情况。在此，我们报告了哺乳动物代谢性生物素化技术的进一步扩展，通过与大肠杆菌的生物素连接酶BirA共分泌，实现对哺乳动物细胞分泌并表达在其细胞表面的蛋白进行生物素化。该技术可用于对分泌蛋白进行生物素化以用于纯化或靶向，也可用于对哺乳动物细胞表面进行生物素化，以促进其与其他未标记细胞的区分和纯化。
