Small protein-domain affinity reagents and D-proteins

抗体是最广泛使用的亲和试剂，其以高亲和力和特异性结合至感兴趣的靶蛋白。抗体一直是研究蛋白质功能及其在正常和疾病状态下细胞信号级联中作用的宝贵工具。此外，这些试剂在诊断和治疗中越来越突出和实用，因为它们可以用于检测或抑制疾病的蛋白质标志物。我的研究项目主要集中在工程小，稳定的蛋白质结构域（从头蛋白质界面，可以潜在地识别目标蛋白质。然而，这些支架的小尺寸在制备良好折叠的亲和试剂方面提出了重大的技术挑战。我们之前已经探讨了设计组合库的几个关键因素。突变界面的结构多样性和氨基酸组成被确定为产生针对多种靶蛋白的亲和试剂的重要因素。*在这个提议中，我们试图研究支架蛋白的结构刚性对产生亲和试剂的有效性的影响。鉴于分子识别需要与靶蛋白的构象匹配，我们假设具有最小构象灵活性的刚性支架可能不是理想的。我们提出了一个系统的研究，通过比较相同的组合库的基础上定期和稳定的版本的给定支架蛋白。我们以前报道了第一次使用小蛋白结构域支架产生折叠的D-蛋白亲和试剂。D-蛋白亲和试剂是稳定的，抗蛋白水解和非免疫原性，使这些试剂在分子成像中的体内应用的理想选择。然而，这是一个新兴的领域，有几个基本问题仍然没有答案。与这些非天然蛋白质相互作用的细胞的命运是什么？这些蛋白质在受体介导的内吞作用后在细胞中积累吗？在这项提案中，我们的目标是通过开发与细胞表面受体EphA 2结合的D-蛋白和L-蛋白亲和试剂来解决这些问题。* 拟议的研究将对开发用于亲和试剂开发的有效组合文库产生影响，并为D蛋白亲和试剂的开发奠定基础。

Antibodies are the most widely used affinity reagents, that bind with high affinity and specificity to a target protein of interest. Antibodies have been valuable tools for studying protein function and their role in cell signaling cascades, in both normal and disease states. In addition, these reagents are gaining prominence and utility in diagnosis and therapy, given that they can be used towards detection or inhibition of protein markers of disease.***My research program focuses on engineering small, stable protein domains (de novo protein interfaces that can potentially recognize target proteins. However, the small size of these scaffolds presents significant technical challenges in making well-folded affinity reagents. We have previously explored several key factors in designing combinatorial libraries. Structural diversity and the amino acid composition of the mutated interface were determined to be important factors in generating affinity reagents to a wide variety of target proteins.***In this proposal, we seek to study the effect of structural rigidity of the scaffold proteins on the effectiveness of generating affinity reagents. Given that molecular recognition requires a conformational fit with the target protein, we hypothesize that rigid scaffolds with minimal conformation flexibility may not be ideal. We propose a systematic study by comparing identical combinatorial libraries based on regular and stabilized versions of a given scaffold protein.***We have previously reported the first use of small protein domain scaffolds to generate folded D-protein affinity reagents. D-protein affinity reagents are stable, resistant to proteolysis and non-immunogenic, making these reagents ideal for in vivo applications in molecular imaging. However, this is a nascent field, with several fundamental questions that remain unanswered. What is the fate of the cells that interact with these unnatural proteins? Do these proteins accumulate in cells following receptor-mediated endocytosis? In this proposal, we aim to address these queries by developing D-protein and L-protein affinity reagents that bind to a cell surface receptor EphA2. ***The proposed studies will have impact in developing effective combinatorial libraries for affinity reagent development and lay the groundwork for development of D-protein affinity reagents.********