Selective Protein Capture by Epitope Imprinting

项目概要/摘要 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。研究 这些分子依赖于我们从复杂的生物混合物中选择性捕获它们的能力。 抗体已被最广泛地用于选择性蛋白质和肽捕获， 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。这些物质在分离方面有重要的应用， 用于生物传感器、毒素中和和生物医学诊断的发展。非- 利用分子印迹的生物学方法在鲁棒网络中产生特异性识别位点 聚合物蛋白质和肽的识别是通过识别目标的暴露结构域（表位）来实现的。 蛋白质，一个独特的九个氨基酸序列。将肽表位用作印迹分子。我们 开发了两种制备用于蛋白质和肽捕获的印迹聚合物的一般方法， 聚合物膜和纳米尺寸印迹聚合物颗粒。印迹膜是通过共价连接 将肽表位固定在玻璃或硅表面上。然后单体在这些表面上聚合， 分子印迹聚合物膜（MIP）。在与官能化表面分离之后，聚合物 评价膜从蛋白质混合物中捕获目标蛋白质的能力。的两种方法 MIP纳米粒子的制备正在发展，沉淀聚合和悬浮 聚合法在这些系统中，在聚合反应中将表位与单体一起引入。 在分离和透析之后，评估纳米颗粒的蛋白质和肽亲和力和特异性。 在两种聚合物形式中，膜和纳米颗粒，捕获在天然条件下实现。 分子印迹是为数不多的用于产生分子受体的通用非生物方法之一。 短表位的选择集中于开发针对肽的一级结构的捕获剂 而不是靶蛋白的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 条件被设计成与天然蛋白质结构相容。它利用了 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 对于模板分子的靶蛋白的一小部分序列，它不使用或需要整个 蛋白因此，这种方法提供了仅基于蛋白质的捕获的机会。 基因组信息

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.