A High Through-put Platform for Recombinant Antibodies to Folded Proteins.
A High Through-put Platform for Recombinant Antibodies to Folded Proteins.
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DOI：
10.1074/mcp.o115.052209
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发表时间：
2015-10
期刊：
影响因子：
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通讯作者：
Wells J
中科院分区：
文献类型：
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作者：
Hornsby M;Paduch M;Miersch S;Sääf A;Matsuguchi T;Lee B;Wypisniak K;Doak A;King D;Usatyuk S;Perry K;Lu V;Thomas W;Luke J;Goodman J;Hoey RJ;Lai D;Griffin C;Li Z;Vizeacoumar FJ;Dong D;Campbell E;Anderson S;Zhong N;Gräslund S;Koide S;Moffat J;Sidhu S;Kossiakoff A;Wells J
抗体是生物学和医学中的关键试剂，但商业来源很少是重组的，因此不能提供永久和可再生的资源。在这里，我们描述了一个工业化的平台，以产生抗原和346个转录因子（TF）和211表观遗传抗原的经验证的重组抗体。我们描述了一个优化的自动化噬菌体展示和抗原表达流水线，总共产生了约3000个测序的抗原结合结构域片段，这些片段具有高亲和力（通常EC 50 <20 nm）、高稳定性（Tm <80 °C）、在E.大肠杆菌（0.5mg/L），以及在复杂细胞裂解物中结合抗原的能力。我们评估了产生同源SCAN结构域的Fab亚组的结合特异性。这些片段抗原结合结构域对其靶SCAN抗原是单特异性的，除了在极少数情况下它们与少数高度相关的抗原交叉反应。值得注意的是，在6个细胞系中对270种TF抗原进行的免疫荧光实验显示，70%的TF抗原主要在细胞质中染色，20%的TF抗原在细胞核中染色，这加强了易位在TF生物学中的主导作用。这些克隆抗体试剂通过我们的网站recombinant-antibodies.org提供给学术界，以允许对TF和染色质生物学进行更系统的分析。我们相信，这些平台、基础设施和自动化方法将在未来十年内为人类蛋白质组的下一代可再生抗体试剂提供便利。
Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade.