Selective Protein Capture by Epitope Imprinting

描述（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，其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稳健合成聚合物。这些物质在分离、用于生物传感器、中和毒素和开发生物医学诊断方面具有重要应用。分子印迹技术是一种非生物学方法，用于在高分子网络中建立特异性识别位点.蛋白质和肽识别通过鉴定靶蛋白的暴露结构域（表位）（独特的九个氨基酸序列）来实现。将肽表位用作印迹分子。我们正在开发两种制备蛋白质和多肽分子印迹聚合物的一般方法，印迹聚合物薄膜和纳米印迹聚合物颗粒。通过将肽表位共价连接到玻璃或硅表面来制备印迹膜。然后单体在这些表面上聚合以产生分子印迹聚合物膜（MIP）。在从功能化表面分离之后，评价聚合物膜从蛋白质混合物中捕获靶蛋白质的能力。两种制备MIP纳米颗粒的方法正在开发中，沉淀聚合和悬浮聚合。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用于肽的一级结构而不是靶蛋白的更复杂的二级和三级结构的捕获剂，并且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相容。它利用基于已知或预测的蛋白质结构的暴露表位的序列。这种方法只需要模板分子的靶蛋白的一小部分的肽序列;它不使用或需要整个蛋白质。因此，这种方法提供了仅基于基因组信息捕获靶蛋白的机会。 公共卫生相关性：抗体是用于生物医学研究、疾病诊断和治疗诸如感染和癌症等疾病的重要试剂。抗体是由细胞系或克隆产生的，这些细胞系或克隆是从已经用作为研究目标的物质免疫的动物中获得的。我们建议开发一种方法，用于生产强大的，廉价的，非生物聚合物抗体，可用作天然抗体的替代品。

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.