Selective Protein Capture by Epitope Imprinting

描述（由申请人提供）：生物大分子如蛋白质和DNA是地球上每一种生命形式所必需的。对这些分子的研究取决于我们从复杂的生物混合物中选择性地捕获它们的能力。抗体已被广泛用于选择性蛋白质和肽捕获，应用于工业蛋白质纯化，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。该提案涉及开发用于选择性捕获肽和蛋白质的强大合成聚合物。这些物质在分离、生物传感器、毒素中和和生物医学诊断方面具有重要应用。分子印迹的非生物学方法被用于在鲁棒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鉴定靶蛋白的暴露结构域（表位）来实现的，这是一个独特的9个氨基酸序列。肽表位被用作印记分子。我们正在开发两种通用的方法来制备用于蛋白质和肽捕获的印迹聚合物，印迹聚合物薄膜和纳米尺寸的印迹聚合物颗粒。印迹膜是通过将肽表位共价附着在玻璃或硅表面来制备的。然后单体在这些表面聚合，产生分子印迹聚合物膜（MIP）。从功能化表面分离后，评估聚合物膜从蛋白质混合物中捕获目标蛋白质的能力。制备MIP纳米粒子的方法有沉淀聚合和悬浮聚合两种。在这些体系中，表位在聚合反应中与单体一起引入。在分离和透析后，纳米颗粒被评估蛋白质和肽的亲和力和特异性。在聚合物格式、薄膜和纳米颗粒中，捕获都是在自然条件下实现的。分子印迹是制造分子受体的几种通用的非生物方法之一。短表位的选择侧重于为肽的一级结构而不是目标蛋白的更复杂的二级和三级结构开发捕获剂，类似于使用肽片段来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。这种方法只需要目标蛋白的一小部分肽序列作为模板分子；它不使用或不需要全蛋白质。因此，这种方法为仅基于基因组信息捕获目标蛋白质提供了机会。

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.