In vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system.
In vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system.
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DOI：
10.1186/1472-6750-8-41
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发表时间：
2008-04-18
影响因子：
3.5
通讯作者：
Burrone OR
中科院分区：
文献类型：
--
作者：
Predonzani A;Arnoldi F;López-Requena A;Burrone OR
由于其极高的强度，生物素与(链)亲和素之间的相互作用已被开发用于大量的生物技术应用。通过在哺乳动物细胞中将目的蛋白与15个氨基酸的生物素受体多肽(BAP)和细菌生物素-蛋白连接酶BIRA共表达，可以实现体内蛋白质的定点生物素化。BIRA可以识别生物素，并将其连接到BAP序列的单个赖氨酸残基上。然而，这个系统主要是基于同时使用两个不同的质粒或通过IRES驱动的机制诱导两种蛋白质的表达。我们开发了一个包含两个独立转录单位的单一双基因质粒，用于BAP标记的蛋白质和Bira酶的工程化版本的共同表达。在编码BIRA的cDNA的上游，添加了一个信号分泌前导序列，以允许酶转位到分泌途径。在免疫荧光显微镜、流式细胞仪和ELISA法中，三种不同形式的重组抗体，膜结合和分泌截短的IgE Fc片段和人IgE高亲和力受体的可溶性版本被有效地生物素化，并保持了它们的结合特性。本研究表明，单一双基因质粒的分泌结合蛋白和膜结合蛋白都具有普遍的适用性，可以诱导标记有短受体多肽的靶分子在体内发生位点特异性的生物素化。这些分子可以很容易地从哺乳动物细胞的上清液或提取物中获得，并用于广泛的生物学应用。
Due to its extremely high strength, the interaction between biotin and (strept)avidin has been exploited for a large number of biotechnological applications. Site-specific biotinylation of proteins in vivo can be achieved by co-expressing in mammalian cells the protein of interest fused to a 15 amino acid long Biotin Acceptor Peptide (BAP) and the bacterial biotin-protein ligase BirA, which specifically recognizes and attaches a biotin to the single lysine residue of the BAP sequence. However, this system is mainly based on the contemporaneous use of two different plasmids or on induction of expression of two proteins through an IRES-driven mechanism. We developed a single bigenic plasmid that contains two independent transcriptional units for the co-expression of both the protein tagged with BAP and an engineered version of the BirA enzyme. Upstream of the cDNA encoding BirA, a signal secretion leader sequence was added to allow translocation of the enzyme to the secretory pathway. Three different recombinant antibodies in the scFv format, a membrane bound and secretory truncated IgE Fc fragment and a soluble version of the human IgE high affinity receptor were shown to be efficiently biotinylated and to maintain their binding properties in immunofluorescence microscopy, flow cytometry and ELISA assays. The present study shows the universal applicability to both secretory and membrane bound proteins of a single bigenic plasmid to induce the site-specific in vivo biotinylation of target molecules tagged with a short acceptor peptide. These molecules could be easily obtained from supernatants or extracts of mammalian cells and used for a wide range of biological applications.
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DOI：
10.1073/pnas.1332608100
发表时间：
2003-06-24
影响因子：
11.1
作者：
de Boer, E;Rodriguez, P;Strouboulis, J
通讯作者：
Strouboulis, J
影响因子：
5.6
作者：
BARKER, DF;CAMPBELL, AM
通讯作者：
CAMPBELL, AM
DOI：
10.1089/153685903322328965
发表时间：
2003-08-01
期刊：
HYBRIDOMA AND HYBRIDOMICS
影响因子：
--
作者：
López-Requena, A;De Acosta, CM;Vázquez, AM
通讯作者：
Vázquez, AM
影响因子：
12.4
作者：
Parrott, MB;Barry, MA
通讯作者：
Barry, MA
影响因子：
3.6
作者：
Lopez-Requena, Alejandro;Bestagno, Marco;Burrone, Oscar R.
通讯作者：
Burrone, Oscar R.