Selective protein capture by epitope imprinting
Selective protein capture by epitope imprinting
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DOI：
10.1002/anie.200503760
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发表时间：
2006-01-01
影响因子：
16.6
通讯作者：
Shea, KJ
中科院分区：
文献类型：
--
作者：
Nishino, H;Huang, CS;Shea, KJ
2452 2006 Wiley-VCH Verlag GmbH&Co.KGaA，Weinheim Angew。化学。2006,118,2452-2456的分子印迹通过在印迹分子存在的情况下通过交联性和功能性单体的聚合在健壮的网络聚合物中产生特定识别位点的群体。蛋白质印迹的成功例子是非常有限和专门的。[8，10-12]在这里，我们报告了一种通过使用分子印迹来创建蛋白质合成受体的一般程序。目标蛋白的一个暴露的结构域(表位)(一个独特的九个氨基酸序列)被用来印记捕获位置。多肽表位以共价方式附着在玻璃或硅表面。然后单体在这些表面聚合，形成分子印迹聚合物(MIP)膜。从功能化表面分离后，聚合物膜结合并捕获来自蛋白质混合物的目标蛋白质。捕获是在当地条件下完成的。生物大分子，如蛋白质和DNA，对地球上的每一种生命形式都是必不可少的。对这些分子的研究一直依赖于我们从复杂的生物混合物中选择性地捕获这些分子的能力。碱基对互补性为选择性地分离和纯化具有所需序列的DNA和RNA分子提供了一种强大而强大的工具。这一工具将继续在分子生物学研究的几乎所有方面发挥作用。抗体已被最广泛地用于选择性捕获蛋白质，并被用于工业蛋白质纯化、基础生物医学研究和临床诊断。然而，抗体表现出的特性限制了它们的应用。这些蛋白质很大(Mr%150K)，是需要小心储存的复杂分子。由于抗体是由活细胞产生的，有时很难控制它们的质量。此外，已知许多蛋白质很难产生抗体。理想的蛋白质捕捉剂应该具有高度的特异性，并由稳定、坚固、非生物的材料组成。分子印迹是创建分子受体的为数不多的一般非生物方法之一。[6]这一策略最成功的应用针对的是低分子量、有机可溶分子。由此产生的分子印迹聚合物(MIP)可以作为药物、环境污染物和其他有毒物质的识别元件。另一方面，多肽和蛋白质靶标提出了更大的挑战，部分原因是它们的结构复杂，而且这些靶标与通常用于印迹的有机溶剂不相容。此外，由于大多数分子印迹聚合物使用目标分子作为模板，因此在使用之前移除所有模板分子是至关重要的。这一担忧可以通过使用不同于目标的模板分子来克服。具体地说，使用来自靶标的表位将消除这一潜在问题。Minoura和他的同事首先展示了这种识别多肽的方法。[13]在这里，我们利用“暴露的”蛋白质表位来印记捕获位置。选择C末端暴露的蛋白质是因为这个位点是翻译后修饰的较不频繁的目标。[14，15]九肽被选为序列长度，因为它代表了用于识别特定蛋白质的非结构域中的几乎唯一的密码。[16，17]短表位的选择侧重于为肽的一级结构开发捕捉剂，而不是为目标蛋白质的更复杂的二级和三级结构开发捕捉剂，如…
2452 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. 2006, 118, 2452–2456 of molecular imprinting creates populations of specific recognition sites in robust network polymers by polymerization of cross-linking and functional monomers in the presence of an imprint molecule.[6, 7] The imprint molecule is either the intended target or some fragment of it. Successful examples of protein imprinting are very limited and specialized.[8, 10–12] Herein we report a general procedure for creating synthetic receptors for proteins by using molecular imprinting. An exposed domain (epitope) of the target protein (a unique nine amino acid sequence) was used to imprint the capture sites. The peptide epitope was covalently attached to a glass or silicon surface. Monomers were then polymerized on these surfaces to produce a molecularly imprinted polymer (MIP) film. Following separation from the functionalized surface, the polymer film bound and captured the target protein from protein mixtures. The capture was achieved under native conditions. Biological macromolecules, such as proteins and DNA, are essential for every life form on earth. Studies of these molecules have been dependent on our ability to selectively capture these molecules from complex biological mixtures. Base-pair complementarity provides a robust and powerful tool for selectively isolating and purifying DNA and RNA molecules with desired sequences. This tool will remain instrumental in virtually all aspects of molecular biology research. Antibodies have been the most widely used for selective protein capture and are used for industrial protein purification, basic biomedical research, and clinical diagnostics. However, antibodies exhibit characteristics that limit their applications. These proteins are large (Mr% 150 K), complex molecules that need to be stored carefully. As antibodies are produced by living cells, it is sometimes difficult to control their quality. Furthermore many proteins are known to be difficult to raise antibodies against. An ideal protein-capture agent should have high specificity and be composed of a stable, robust, nonbiological material. Molecular imprinting is one of the few general, nonbiological methods for creating molecular receptors.[6] Most successful applications of this strategy target low molecular weight, organic-soluble molecules. The resulting molecularly imprinted polymers (MIPs) can function as the recognition element for drugs, environmental pollutants, and other toxic substances. Peptide and protein targets, on the other hand, present greater challenges due, in part, to their structural complexity and the incompatibility of these targets with organic solvents that are typically used for imprinting. Furthermore, as most molecularly imprinted polymers use target molecules as templates, it is critical to remove all template molecules before their use. This concern can be overcome by the use of template molecules that differ from the target. Specifically, the use of an epitope from the target would eliminate this potential problem. Minoura and coworkers first demonstrated this approach for peptide recognition.[13] Herein, we utilize “exposed” epitopes of proteins to imprint the capture site. Proteins with exposed C-termini were selected as this site is a less frequent target for posttranslational modification.[14, 15] Nonapeptides were chosen as the sequence length as it represents a near-unique code in unstructured domains for the identification of a specific protein.[16, 17] 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 structures of a target proteins …