Secrete, Capture, Sort, Sequence: NGS Decoded Molecular Recognition Pairs

尽管基于单抗的技术占主导地位，但通过单抗 抗体的产生和传播，连同免疫球蛋白的大小和结构，施加了基本的 对其有用性的限制，特别是用于研究目的。糟糕的专用性和文档记录导致 实验重现性方面的危机，导致美国每年浪费约3.5亿美元的研究支出。 通过系统地产生经测序的、有效的单克隆来纠正这一点将花费估计 每种抗体50,000美元，许多靶点都会失败。 单克隆体的局限性将通过发展酵母菌‘分泌和捕获’共同展示来解决 重组非免疫纳米抗体(NBS)的高通量分离和改良系统 来源于骆驼抗体VHH结构域的蛋白质亲和试剂。基于荧光素的FACS技术 量化显示的NBS和捕获的目标蛋白结构域(TPD)将与下一代集成 确定相关复合体的测序(NGS)。整合将极大地扩展曲目 可为其分离同源NBs的生物目标，并促进创建有重点的NB工具包。 目前的非免疫支架筛选使用纯化的目标蛋白来分离候选结合蛋白，这些结合蛋白 物理克隆和单独评估。这一资源密集型方法将被以下内容所取代： (1)定量报道共表达的Nb和TPDs在 在特异性、亲和力和动力学方面，因此避免使用纯化的蛋白质。这个测试是基于融合 表面展示的NBS和分泌的TPD以光谱方式区分的氟原激活蛋白(FAP) 当结合非荧光染料(荧光素)时发出荧光；荧光素可以通过聚乙二醇灵活地连接成“扎染染料”。 一种可切割的扎染染料用于稳定和报告分泌的TPD和同源物的细胞表面复合体 显示的Nb；在切割时，从细胞表面释放TPD-FAP的动力学分析允许人们估计 Nb/Tpd亲和力。 (2)物理融合共表达的NB和TPD的编码基因类型的方法，从而实现NGS 分析将复杂群体的FACS分析分解为单个克隆的结合表型，从而 消除了物理克隆的需要。分别编码NBS和NBS的酵母文库大规模交配后 CRISPR将被用来强制编码序列的批量重组 NB和条形码，该条形码将相关联的TPD标识为单个NGS可解码读取帧。 (3)生物信息学派生的TPD查询集形式的多路筛选，以直接获得组 相关试剂，从而最大限度地减少了对单个克隆进行连续评估的需要。将使用查询来隔离 探索以前很难接近的生物功能的NBS；我们的测试案例将是：(I) 神经连接蛋白剪接异构体；(Ii)神经连接蛋白/神经毒素复合体；以及(Iii)细菌表面蛋白胞外结构域。

Despite the predominance of monoclonal antibody based technologies, the means by which monoclonal antibodies are generated and disseminated, together with the size and structure of IgGs, impose fundamental limitations on their usefulness, especially for research purposes. Poor specificity and documentation has led to a crisis in experimental reproducibility, leading to an annual waste of ~$350 million in US research expenditures. Rectifying this by the systematic generation of sequenced, validated monoclonals would cost an estimated $50,000 per antibody and would fail for many targets. Limitations of monoclonals will be addressed by developing a yeast `secrete and capture' co-display system for the high throughput isolation and improvement of recombinant nonimmune Nanobodies (NBs), small protein affinity reagents derived from camelid antibody VHH domains. Fluorogen-based FACS technology that quantifies displayed NBs and captured target protein domains (TPDs) will be integrated with next generation sequencing (NGS) that identifies the associated complex. Integration will greatly expand the repertoire of biological targets for which cognate NBs may be isolated, and facilitate the creation of focused NB toolkits. Current nonimmune scaffold screens use purified target protein to isolate candidate binders that are physically cloned and individually evaluated. This resource-intensive approach will be replaced by the following: (1) A FACS reporter assay that quantitatively reports the interaction of co-expressed NB and TPDs in terms of specificity, affinity and kinetics, thus avoiding the use of purified protein. The assay is based on fusing surface-displayed NBs and secreted TPDs to spectrally distinct fluorogen activating proteins (FAPs) that fluoresce when binding non-fluorescent dyes (fluorogens); fluorogens may flexibly linked by PEG as a `tie-dye'. A cleavable tie-dye is used to stabilize and report on a cell surface complex of secreted TPD and cognate displayed NB; upon cleavage, kinetic analysis of TPD-FAP release from the cell surface allows one to estimate NB/TPD affinity. (2) A method that physically fuses the encoded genotypes of co-expressed NB and TPD, enabling NGS analysis to resolve FACS assays of complex populations into the binding phenotypes of individual clones, thus eliminating the need for physical cloning. After mass mating of yeast libraries respectively encoding NBs and secreted TPDs on separate plasmids, CRISPR will be used to force bulk recombination of sequences encoding the NB and a barcode that identifies the associated TPD into a single NGS decodable read frame. (3) Multiplexed screens in the form of bioinformatics derived TPD query sets to directly obtain groups of related reagents, thus minimizing the need to serially evaluate individual clones. Queries will be used to isolate NBs that probe biological functionalities that were previously very difficult to approach; our test cases will be: (i) neuroligin splice isoforms; (ii) neuroligin/neurexin complexes; and (iii) bacterial surface protein ectodomains.