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

分泌、捕获、排序、测序：NGS 解码的分子识别对

• 批准号: 9467841
• 负责人: Marcel P Bruchez
• 金额: $ 31.62万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9467841
• 关键词: Address; Affinity; Alpha Cell; Antibodies; Binding; Bioinformatics; Biological; Biological Assay; Biological Models; Biological Process; Biology; Cell Separation; Cell surface; Cells; Cleaved cell; Cloning; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Color; Complex; DNA Sequence; Dissociation; Documentation; Dyes; Electron Transport Complex III; Expenditure; Generations; Genetic Recombination; Genotype; Hereditary Disease; Immunoglobulin G; Individual; Kinetics; Libraries; Link; Mediating; Medicine; Membrane Proteins; Methods; Monitor; Monoclonal Antibodies; Mus; Neurons; Output; Partner in relationship; Patient Self-Report; Phenotype; Plasmids; Population; Population Heterogeneity; Protein Binding Domain; Protein Isoforms; Proteins; Proxy; RNA Splicing; Reagent; Recombinant Proteins; Recombinants; Reporter; Reporting; Reproducibility; Research; Resources; Specific qualifier value; Specificity; Staphylococcus aureus; Streptococcus pneumoniae; Structure; Surface; Synapses; System; Technology; Tertiary Protein Structure; Testing; Work; Yeasts; base; combinatorial; cost; design; flexibility; high throughput technology; homologous recombination; interest; member; molecular recognition; nanobodies; next generation sequencing; novel; protein complex; scaffold; screening; technology development; wasting

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.

尽管以单克隆抗体为基础的技术占主导地位，单克隆的手段