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首页> 外文期刊>Biomolecular engineering >Metabolic biotinylation of recombinant antibody by biotin ligase retained in the endoplasmic reticulum
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Metabolic biotinylation of recombinant antibody by biotin ligase retained in the endoplasmic reticulum

机译：内质网中保留的生物素连接酶对重组抗体的代谢生物素化

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摘要

Due to its strength and specificity, the interaction between avidin and biotin has been used in a variety of scientific and medical applications ranging from immumohistochemistry to drug targeting. The present study describes two methods for biotinylation of proteins secreted from eukaryotic cells using the Escherichia coli biotin protein ligase. In one system the biotin ligase was co-secreted from the cells along with substrate protein enabling extracellular biotinylation of the tagged protein. In the other system, biotin ligase was engineered to be retained in the endoplasmic reticulum (ER) and metabolically biotinylates the secretory protein as it passes through the ER. An engineered antibody fragment, a diabody with specificity for carcinoembryonic antigen (CEA) was fused to the biotin acceptor domain (123 amino acid) of Propionibacterium shermanii. Coexpression of the fusion protein with ER retained biotin ligase showed higher biotinylation efficiency than biotinylation by co-secreted ligase. Biotinylation of the anti-CEA diabody tagged with a short (15 amino acid, Biotin Avitag (TM)) biotin acceptor peptide was also successful. Utilization of ER retained biotin ligase for biotinylation of protein is an attractive alternative for efficiently producing uniformly biotinylated recombinant proteins for a variety of avidin-biotin technologies. (C) 2007 Elsevier B.V. All rights reserved.
机译：由于其强度和特异性，抗生物素蛋白和生物素之间的相互作用已用于从免疫组织化学到药物靶向的多种科学和医学应用中。本研究描述了两种使用大肠杆菌生物素蛋白质连接酶对真核细胞分泌的蛋白质进行生物素化的方法。在一个系统中，生物素连接酶与底物蛋白一起从细胞中共同分泌，从而使标记蛋白能够进行细胞外生物素化。在另一个系统中，生物素连接酶经过工程改造后可保留在内质网（ER）中，并在分泌蛋白通过ER时对其进行代谢生物素化。将工程改造的抗体片段（对癌胚抗原（CEA）具有特异性的双抗体）融合至谢尔曼丙酸杆菌的生物素受体域（123个氨基酸）。融合蛋白与ER保留的生物素连接酶的共表达显示出比共分泌连接酶的生物素化更高的生物素化效率。用短的（15个氨基酸，Biotin Avitag（TM））生物素受体肽标记的抗CEA双抗体的生物素化也成功。利用ER保留的生物素连接酶进行蛋白质的生物素化是一种吸引人的选择，可以有效地为各种抗生物素蛋白-生物素技术生产均匀生物素化的重组蛋白。 （C）2007 Elsevier B.V.保留所有权利。

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