Selective Protein Capture by Epitope Imprinting

项目摘要/摘要 蛋白质和DNA等生物大分子对地球上每一种生命形式都是必不可少的。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地捕获它们的能力。 抗体已被最广泛地用于选择性捕获蛋白质和多肽，应用于 工业蛋白质提纯、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。该提案涉及到开发强健的合成聚合物。 用于选择性捕获多肽和蛋白质。这些物质在分离方面有重要的应用， 用于生物传感器、毒素的中和和生物医学诊断的发展。非-- 分子印迹的生物学方法被用来在健壮网络中创建特定的识别位点 聚合物。蛋白质和多肽的识别是通过识别目标的暴露区域(表位)来实现的 蛋白质，一个独特的九个氨基酸序列。该多肽表位被用作印迹分子。我们是 发展两种制备用于蛋白质和肽捕获的印迹聚合物的通用方法 聚合物薄膜和纳米尺寸的印迹聚合物颗粒。印迹薄膜是通过共价连接来制备的 将多肽表位连接到玻璃或硅表面。然后在这些表面上聚合单体以生产 分子印迹聚合物膜(MIP)。在从官能化表面分离后，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。有两种方法可用于 MIP纳米粒子的制备正在开发中，沉淀聚合和悬浮 聚合反应。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 而不是靶蛋白的更复杂的二级和三级结构，并且类似于 产生表位选择性抗体的多肽片段和合成材料。此外，抓捕 设计的条件与天然蛋白质结构兼容。它利用以下序列 基于已知或预测的蛋白质结构的暴露表位。这种方法只需要多肽 一小部分靶蛋白的序列为模板分子，它不使用或需要完整 蛋白。因此，这种方法提供了捕获目标蛋白的机会，仅基于 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.