A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry
A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry
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DOI：
10.1093/protein/gzm090
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发表时间：
2008-04-01
影响因子：
2.4
通讯作者：
Stahl, Stefan
中科院分区：
文献类型：
--
作者：
Kronqvist, Nina;Lofblom, John;Stahl, Stefan
在这里，我们描述了首次报道使用革兰氏阳性细菌系统从展示在肉色葡萄球菌表面的大型组合文库中选择亲和蛋白。基于葡萄球菌蛋白A的58个残基结构域的3×10(9)变异体文库，利用一个周期的噬菌体展示预富集与人肿瘤坏死因子-α(TNF-α)结合，然后转移到葡萄球菌宿主(类似于10(6)变异体)。葡萄球菌展示文库经过三轮流式细胞仪分选，筛选出的克隆通过细胞内分析进行与肿瘤坏死因子-α的结合排序，并通过生物传感器分析和圆二色谱进一步表征。成功的分选产生了三种不同的高亲和力结合蛋白(范围从95 PM到2.2 NM)，并构成了使用革兰氏阳性细菌展示首次选择一种新的亲和蛋白。该方法结合了与细菌宿主合作的简单性和在强大的革兰氏阳性细菌上展示重组蛋白的优势，以及在选择和表征过程中使用强大的流式细胞术。
Here we describe the first reported use of a Gram-positive bacterial system for the selection of affinity proteins from large combinatorial libraries displayed on the surface of Staphylococcus carnosus. An affibody library of 3 x 10(9) variants, based on a 58 residue domain from staphylococcal protein A, was pre-enriched for binding to human tumor necrosis factor-alpha (TNF-alpha) using one cycle of phage display and thereafter transferred to the staphylococcal host (similar to 10(6) variants). The staphylococcal-displayed library was subjected to three rounds of flow-cytometric sorting, and the selected clones were screened and ranked by on-cell analysis for binding to TNF-alpha and further characterized using biosensor analysis and circular dichroism spectroscopy. The successful sorting yielded three different high-affinity binders (ranging from 95 pM to 2.2 nM) and constitutes the first selection of a novel affinity protein using Gram-positive bacterial display. The method combines the simplicity of working with a bacterial host with the advantages of displaying recombinant proteins on robust Gram-positive bacteria as well as using powerful flow cytometry in the selection and characterization process.