An Efficient Phage Display Based Protease Activity Profiling Platform

基于高效噬菌体展示的蛋白酶活性分析平台

• 批准号: 10412127
• 负责人: Harry Benjamin Larman
• 金额: $ 20.47万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10412127
• 关键词: Affinity; Antibody Specificity; Apoptotic; Automation; Bacteriophage T7; Bacteriophages; Binding; Biochemical; Biological; Biological Assay; Biological Process; Biotin; Blood; C-terminal; Cancer Biology; Caspase; Cell Culture Techniques; Cells; Complex; Coupled; DNA; Data; Data Analyses; Databases; Development; Dictionary; Digestion; Down-Regulation; Endopeptidases; Enzymes; Epitopes; Escherichia coli; Excision; Frequencies; Generations; Growth; Human; Immobilization; Immunology; Individual; Isopropyl Thiogalactoside; Knowledge; Libraries; Life; Malignant Neoplasms; Measures; Metalloproteases; Methods; Molecular; Multienzyme Complexes; Myeloid Cells; N-terminal; Names; Oligonucleotides; Pathway interactions; Peptide Hydrolases; Peptides; Performance; Phage Display; Phage ImmunoPrecipitation Sequencing; Phase; Physiological; Process; Prodrugs; Promoter Regions; Proteolysis; Proteome; Proteomics; Protocols documentation; Recombinants; Reproducibility; Research Personnel; Resolution; Role; Sampling; Solid; Specimen; Streptavidin; System; Techniques; Technology; Testing; Tissues; Tumor Biology; Up-Regulation; Urine; Work; anticancer research; base; cancer cell; design; detection limit; enzyme substrate; experimental study; flexibility; human tissue; improved; insight; interest; magnetic beads; neoplastic cell; novel; screening; therapeutic candidate; therapeutic target; tumor; tumor immunology; tumor-immune system interactions; vector

PROJECT SUMMARY/ABSTRACT Protein cleavage is a tightly regulated biological process, which is often dysfunctional in cancer. The lack of scalable, unbiased assays has severely limited our ability to characterize the spectrum of protease substrates, or to measure proteolytic activities within tumor specimens, blood or urine samples. Here we propose to develop an efficient phage display based protease activity profiling platform to enable the sensitive and unbiased analysis of protease activities associated with human cancers. In preliminary studies, we demonstrate how a novel phage display vector, coupled with a complete human ‘peptidome’ phage library, can be used to efficiently profile proteases. We have named this approach Sensing EndoPeptidase Activities via RemovAl of Tag Epitopes, or ‘SEPARATE’. Initial experiments indicate that substrate cleavage can be robustly detected when proteases are profiled at physiological concentrations, and that novel biological insight can arise from such analyses. Here we propose to develop workflows and analytical pipelines for unbiased SEPARATE profiling of (i) recombinant proteases, (ii) complex biological samples, and (iii) genetically encoded proteases. We expect that process automation and sample multiplexing will enable highly reproducible, inexpensive (~$40 per sample), proteome-wide profiling of protease activities. Aim 1: Establish ‘solid phase’ SEPARATE for single enzymes and biological samples. A flexible, optimized workflow will be developed to analyze recombinant proteases and complex biological specimens. Using proteases from different catalytic classes, we will optimize the SEPARATE assay conditions and determine assay performance metrics. The utility of the platform will be tested by validating novel protease substrates of relevance to cancer biology/immunology. Aim 2: Establish ‘co-expression’ SEPARATE for genetically-encoded proteases. Most proteases are not commercially available as active enzymes. The ability to profile genetically encoded proteases in high throughput would enable generation of a large protease-substrate database, ultimately facilitating the deconvolution of complex SEPARATE data. A system will be established to express recombinant proteases inside E. coli host cells only during active replication of the human peptidome library. Methods of separating phages displaying cleaved peptides from phages displaying uncleaved peptides will be developed. Data from the analysis of complex biological samples will be deconvoluted by means of a large protease-peptide lookup table. Impact: This project will provide cancer researchers with the ability to comprehensively profile individual proteases, as well as complex cancer-relevant biological samples in high throughput.

项目概要/摘要 蛋白质裂解是一个受到严格调控的生物过程，在癌症中通常会出现功能障碍。 缺乏可扩展、公正的检测方法严重限制了我们表征蛋白酶谱的能力 底物，或测量肿瘤标本、血液或尿液样本中的蛋白水解活性。 我们建议开发一种基于噬菌体展示的高效蛋白酶活性分析平台，以实现 对与人类癌症相关的蛋白酶活性进行灵敏且公正的分析。 在初步研究中，我们展示了新型噬菌体展示载体如何与完整的噬菌体展示载体相结合。 人类“肽组”噬菌体库，可用于有效地分析蛋白酶，我们将其命名为。 通过去除标签表位或“单独的初始”来检测内切肽酶活性。 实验表明，当蛋白酶在以下位置进行分析时，可以可靠地检测到底物裂解 生理浓度，并且可以从此类分析中产生新颖的生物学见解。 在这里，我们建议开发工作流程和分析管道，以进行公正的单独分析 (i) 重组蛋白酶，(ii) 复杂的生物样品，以及 (iii) 基因编码的蛋白酶。 我们期望过程自动化和样品复用将实现高度可重复、 价格低廉（每个样品约 40 美元），对蛋白酶活性进行全蛋白质组分析。 目标 1：建立单一酶和生物样品的“固相”分离。 将开发灵活、优化的工作流程来分析重组蛋白酶和复杂的酶 生物样本。使用不同催化类别的蛋白酶，我们将优化单独的酶。 分析条件并确定分析性能指标将通过以下方式进行测试。 验证与癌症生物学/免疫学相关的新型蛋白酶底物。 目标 2：为基因编码的蛋白酶建立单独的“共表达”。 大多数蛋白酶不能作为活性酶在商业上获得。 能够进行基因分析。 高通量编码的蛋白酶将能够生成大型蛋白酶底物数据库， 最终将建立一个系统来促进复杂的单独数据的反卷积。 仅在人类活跃复制期间在大肠杆菌宿主细胞内表达重组蛋白酶 将展示切割肽的噬菌体与展示噬菌体的方法分离。 将开发来自复杂生物样品分析的数据。 通过大型蛋白酶肽查找表进行解卷积。 影响：该项目将为癌症研究人员提供全面分析的能力 高通量分析单个蛋白酶以及复杂的癌症相关生物样品。