Selective Protein Capture by Epitope Imprinting

通过表位印迹选择性蛋白质捕获

• 批准号: 8008962
• 负责人: KENNETH J SHEA
• 金额: $ 4.03万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-28 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8008962
• 关键词: Affinity; Amino Acid Sequence; Amino Acids; Animals; Antibodies; Antibody-Producing Cells; Binding; Bioinformatics; Biological; Biological Markers; Biomedical Research; Biosensor; Buffers; Cell Line; Characteristics; Cherry - dietary; Clinical; Collaborations; Complex; DNA; Development; Diagnostic; Dialysis procedure; Disease; Enzymes; Epitopes; Exhibits; Film; Genomics; Glass; Goals; Immunoglobulin G; In Vitro; Infection; Institutes; Lead; Length; Life; Malignant Neoplasms; Methods; Molecular; Peptide Fragments; Peptide Receptor; Peptides; Performance; Plasma; Plastics; Polymers; Precipitation; Preparation; Protein Binding; Protein Structure Databases; Proteins; Quartz; Reaction; Reagent; Research; Silicon; Site; Solutions; Specificity; Structure; Surface; Suspension substance; Suspensions; System; Techniques; Tertiary Protein Structure; Therapeutic; Toxin; abstracting; analytical tool; base; cost; design; disease diagnosis; imprint; macromolecule; monomer; nanoparticle; nanosized; particle; peptide structure; polymerization; professor; programs; protein aminoacid sequence; protein purification; protein structure; receptor

Project Summary/Abstract Biological macromolecules such as proteins and DNA are essential for every life form on earth. Studies of these molecules are dependent on our ability to selectively capture them from complex biological mixtures. Antibodies have been the most widely used for selective protein and peptide capture with applications for industrial protein purification, basic biomedical research and in clinical diagnostics. Antibodies however exhibit characteristics that limit their applications. This proposal is involved with developing robust synthetic polymers for selective capture of peptides and proteins. These substances have important applications for separations, for use in biosensors, neutralization of toxins and for the development of biomedical diagnostics. The non- biological approach of molecular imprinting is used to create specific recognition sites in robust network polymers. Protein and peptide recognition is achieved by identifying an exposed domain (epitope) of the target protein, a unique nine amino acid sequence. The peptide epitope is used as the imprint molecule. We are developing two general methods for preparing imprinted polymers for protein and peptide capture, imprinted polymer films and nanosize imprinted polymer particles. Imprinted films are prepared by covalently attaching the peptide epitope to a glass or silicon surface. Monomers are then polymerized on these surfaces to produce a molecularly imprinted polymer film (MIP). Following separation from the functionalized surface, the polymer film is evaluated for its ability to capture the target protein from protein mixtures. Two methods for the preparation of MIP nanoparticles are being developed, precipitation polymerization and suspension polymerization. In these systems, epitopes are introduced with monomers in the polymerization reaction. Following isolation and dialysis, the nanoparticles are evaluated for protein and peptide affinity and specificity. In both polymer formats, films and nanoparticles, the capture is achieved under native conditions. Molecular imprinting is one of the few general, non-biological methods for creating molecular receptors. The choice of short epitopes focuses on developing capture agents for the primary structure of the peptide rather than the more complex secondary and tertiary structure of a target protein and is similar to the use of peptide fragments to generate epitope selective antibodies and synthetic materials. In addition, the capture conditions were designed to be compatible with the native protein structure. It utilizes the sequences of exposed epitopes based on known or predicted protein structure. This method requires only the peptide sequence of a small portion of the target protein for the template molecule, it does not use or need whole protein. As such, this approach provides opportunities for the capture of target proteins based only on genomic information.

项目概要/摘要 蛋白质和 DNA 等生物大分子对于地球上的每种生命形式都是必不可少的。研究 这些分子取决于我们从复杂的生物混合物中选择性捕获它们的能力。 抗体是最广泛用于选择性蛋白质和肽捕获的应用 工业蛋白质纯化、基础生物医学研究和临床诊断。然而抗体表现出 限制其应用的特性。该提案涉及开发坚固的合成聚合物 用于选择性捕获肽和蛋白质。这些物质在分离、 用于生物传感器、中和毒素以及用于生物医学诊断的开发。非 分子印记的生物学方法用于在强大的网络中创建特定的识别位点 聚合物。蛋白质和肽识别是通过识别目标的暴露域（表位）来实现的 蛋白质，独特的九个氨基酸序列。肽表位用作印记分子。我们是 开发两种制备用于蛋白质和肽捕获的印迹聚合物的通用方法，印迹 聚合物薄膜和纳米尺寸印迹聚合物颗粒。压印薄膜是通过共价连接制备的 将肽表位附着到玻璃或硅表面。然后单体在这些表面上聚合以产生 分子印迹聚合物薄膜（MIP）。从功能化表面分离后，聚合物 评估薄膜从蛋白质混合物中捕获目标蛋白质的能力。两种方法可用于 正在开发MIP纳米颗粒的制备，沉淀聚合和悬浮聚合 聚合。在这些系统中，表位在聚合反应中与单体一起引入。 分离和透析后，评估纳米颗粒的蛋白质和肽亲和力和特异性。 在聚合物形式、薄膜和纳米颗粒中，捕获都是在自然条件下实现的。 分子印迹是创建分子受体的少数通用非生物方法之一。 短表位的选择侧重于开发肽一级结构的捕获剂 而不是目标蛋白更复杂的二级和三级结构，并且类似于使用 肽片段以产生表位选择性抗体和合成材料。此外，捕获 条件被设计为与天然蛋白质结构兼容。它利用了以下序列 基于已知或预测的蛋白质结构的暴露表位。该方法仅需要肽 模板分子的目标蛋白的一小部分序列，它不使用或不需要完整的 蛋白质。因此，这种方法为仅基于 基因组信息。