Minimal Protein Affinity Tags

项目摘要 生物医学在很大程度上依赖于将配体结合位点连接到蛋白质的方法。这种亲和标签 对于蛋白质亲和纯化、改善蛋白质溶解度和增加功能性是必需的， 蛋白质如荧光标记或化学修饰位点。该研究旨在开发 使用仅包含1-2个氨基酸残基的亲和标签修饰靶蛋白的一般方法。 最小亲和力标签通过小的（1- 2kDa）合成的寡核苷酸以高亲和力位点特异性地结合。 受体，可以结合到固体支持物和小分子。受体是稳定的和功能性的 在生理条件下，包括血清中，与这些受体结合的蛋白质可以容易地释放， 小分子竞争者的加入。提出的目标包括：1）进一步优化二肽 识别位点; 2）研究合成受体与蛋白质中已经存在的靶位点的结合 3）将最小亲和标签安装到重组蛋白中; 蛋白质和蛋白质药物;和4）开发合成受体的功能衍生物， 为目标蛋白质带来功能性。这样的最小亲和性标签将为靶分子添加实质性功能。 蛋白质，同时对其结构和活性的影响最小。拟议的工作建立在规则之上， 用于通过葫芦[N]脲选择性地、高亲和力识别肽和蛋白质， PI的本科生研究实验室。许多研究小组已经开发了基于这些规则的应用程序， 我们预计，拟议的研究将建立一个核心能力，应促进现有的 生物医学应用并激发新的应用。

PROJECT SUMMARY Biomedical science relies heavily on methods for attaching ligand binding sites to proteins. Such affinity tags are essential for protein affinity purification, for improving protein solubility, and for adding functionality to proteins such as fluorescent labels or sites for chemical modification. The proposed research aims to develop a general method for modifying target proteins using affinity tags that comprise only 1-2 amino acid residues. The minimal affinity tags are bound site-specifically and with high-affinity by small (1-2 kDa) synthetic receptor that can be conjugated to solid supports and small molecules. The receptors are stable and functional in physiologic conditions, including serum, and proteins bound to these receptors can be released easily upon the addition of small molecule competitors. The proposed aims include: 1) further optimizing dipeptide recognition sites; 2) investigating the binding of synthetic receptors to target sites that already exist in protein pharmaceuticals and other bioactive peptides and proteins; 3) installing minimal affinity tags into recombinant proteins and protein pharmaceuticals; and 4) developing functional derivatives of the synthetic receptor to bring functionality to the target proteins. Such minimal affinity tags will add substantial function to target proteins while having minimal impact on their structure and activity. The proposed work builds on the rules for selective, high-affinity recognition of peptides and proteins by cucurbit[n]urils established in the undergraduate research lab of the PI. Many research groups have developed applications built on these rules, and we anticipate that the proposed research will establish a core competency that should facilitate existing biomedical applications and inspire new ones.