Selective Protein Capture by Epitope Imprinting

通过表位印迹选择性蛋白质捕获

• 批准号: 8016626
• 负责人: KENNETH J SHEA
• 金额: $ 23.43万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8016626
• 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; 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; 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; public health relevance; receptor

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Antibodies are important reagents that are used in biomedical research, in diagnosis of diseases, and in treatment of such diseases as infections and cancer. Antibodies are produced by cell lines or clones obtained from animals that have been immunized with the substance that is the target of study. We propose to develop a method for producing robust, inexpensive, non-biological polymer antibodies that can be used as substitutes for native antibodies.

描述(申请人提供)：蛋白质和DNA等生物大分子是地球上每一种生命形式所必需的。对这些分子的研究依赖于我们从复杂的生物混合物中选择性地捕获它们的能力。抗体在工业蛋白质纯化、基础生物医学研究和临床诊断中应用最广泛，可用于选择性捕获蛋白质和多肽。然而，抗体表现出的特性限制了它们的应用。这项建议涉及开发用于选择性捕获多肽和蛋白质的坚固的合成聚合物。这些物质在分离、用于生物传感器、中和毒素和发展生物医学诊断学方面有着重要的应用。分子印迹的非生物方法被用来在健壮的网络聚合物中创建特定的识别位点。蛋白质和肽的识别是通过识别目标蛋白质的暴露区域(表位)实现的，这是一个独特的九个氨基酸序列。该多肽表位被用作印迹分子。我们正在开发两种制备用于捕获蛋白质和多肽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。印迹膜是通过将多肽表位共价连接到玻璃或硅表面来制备的。然后单体在这些表面聚合，形成分子印迹聚合物膜(MIP)。从功能化表面分离后，对聚合物膜从蛋白质混合物中捕获目标蛋白质的能力进行评估。目前制备MIP纳米粒子的方法有两种：沉淀法和悬浮聚合法。在这些体系中，表位与单体一起引入聚合反应。在分离和透析后，对纳米颗粒的蛋白质和多肽的亲和力和特异性进行评估。在两种聚合物形式、薄膜和纳米颗粒中，捕获都是在自然条件下实现的。分子印迹是产生分子受体的为数不多的一般非生物方法之一。短表位的选择侧重于为多肽的一级结构开发捕捉剂，而不是目标蛋白质更复杂的二级和三级结构，类似于使用多肽片段来产生表位选择性抗体和合成材料。此外，捕获条件的设计与天然蛋白质结构兼容。它利用基于已知或预测的蛋白质结构的暴露表位序列。这种方法只需要模板分子的一小部分目标蛋白质的肽序列；它不使用或不需要整个蛋白质。因此，这种方法提供了仅基于基因组信息捕获目标蛋白质的机会。 公共卫生相关性：抗体是重要的试剂，用于生物医学研究、疾病诊断以及感染和癌症等疾病的治疗。抗体是由从动物身上获得的细胞系或克隆产生的，这些动物已经用作为研究目标的物质进行了免疫。我们建议开发一种方法来生产强大的、廉价的、非生物的聚合物抗体，可以用作天然抗体的替代品。