Specific binder for Lightning-Link® biotinylated proteins from an antibody phage library.
Specific binder for Lightning-Link® biotinylated proteins from an antibody phage library.
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DOI：
10.1016/j.jim.2013.06.010
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发表时间：
2013-09-30
影响因子：
2.2
通讯作者：
Bradbury, Andrew R. M.
中科院分区：
文献类型：
--
作者：
Ferrara, Fortunato;Naranjo, Leslie A.;D'Angelo, Sara;Kiss, Csaba;Bradbury, Andrew R. M.
许多应用需要蛋白质生物素化。我们通常使用生物素化蛋白从噬菌体和/或酵母展示文库中选择单链抗体。在噬菌体选择过程中，生物素化抗原与链霉亲和素偶联磁珠结合，而在酵母展示过程中，生物素化抗原在流式细胞术中用于分析和分选。Lightning-Link®生物素试剂盒是一种快速直接的生物素化试剂盒，避免了透析的需要，在可用蛋白质的量有限时特别有用。在抗体文库的常规筛选中，我们发现了一个特异性克隆，该克隆结合了一个通用的新表位，该表位仅在抗原使用商业Lightning-Link®试剂盒生物素化时产生，亲和力为~10nM。非生物素化蛋白，以及那些使用替代方法（赛默飞世尔商业试剂盒或使用Avitag的体内生物素化）进行生物素化的蛋白，不被该抗体识别。利用深度序列分析，该特异性抗体在许多不同的选择中被鉴定为最丰富的。这表明在使用这种修饰试剂时需要谨慎，因为可能会选择针对修饰的抗体，而不是目标蛋白，并且也突出了在基于显示的选择过程中深度测序分析的价值。此外，该抗体在用该方法分析生物素化蛋白方面可能具有很大的实用性。
Many applications required protein biotinylation. We routinely use biotinylated proteins to select single chain antibodies from phage and/or yeast display libraries. During phage selection the biotinylated antigens are bound to streptavidin coupled magnetic beads, while during yeast display, the biotinylated antigens are used during flow cytometry for both analysis and sorting. The Lightning-Link® Biotin kit, a rapid straightforward biotinylation kit that avoids the need for dialysis, is particularly useful when the amount of available protein is limiting. During routine screening of antibody libraries we identified a specific clone that bound a universal neo-epitope generated only when antigens are biotinylated with the commercial Lightning-Link®kit, with an affinity of ~10nM. Non-biotinylated proteins, and those biotinylated using alternative methods – the Thermo Fisher commercial kit or in vivo biotinylation using the Avitag –, were not recognized by this antibody. Using deep sequence analysis, the specific antibody was identified as being the most abundant in a number of different selections. This indicates the need for caution when using such modifying reagents, because of the possibility of selecting antibodies against the modification, rather than the target protein, and also highlights the value of deep sequencing analysis during display based selections. Furthermore, this antibody may have great utility in the analysis of proteins biotinylated using this method.
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