Collaborative Research: Towards a General Design Approach to Arrest Non-Native Aggregation of Multi-Domain Proteins

在整个生物技术和生物制药行业，非原生聚集是成功过表达和纯化重组蛋白的普遍障碍。对于多结构域蛋白，如天然抗体和设计抗体，以及其他主要的β蛋白，情况尤其如此。从根本上说，由于对控制多结构域蛋白质聚集的序列和结构的关键特征的机制理解的限制，这些更复杂蛋白质的合理设计受到阻碍。该项目旨在为填补对多结构域蛋白质聚集机制的基本理解的空白奠定基础，并提供第一代设计规则来增强聚集抗性。人γ - d晶体蛋白（γ D-Crys）将成为模型多结构域蛋白，选择它是因为它具有许多理想的特征：(1)它是单链，双结构域蛋白；(2)它代表了一组重要的全β蛋白；(3)已形成聚集和折叠行为；(4)它有可用的、易于聚集的突变体。更广泛的影响-拟议的研究将导致对多域，全β希腊关键蛋白的聚集和聚集抗性的合理设计的机制理解的改进，其中许多抗体结构是广泛的生物和生物技术兴趣的一个亚类。该研究还将为pi实验室的研究生和本科生的教育和培训提供一个框架，特别关注尖端的实验和建模工具。和过去一样，pi将涉及来自科学和工程领域代表性不足群体的学生。最后，将提出一系列的例子和问题，将本研究的各个方面纳入本科课程，包括计算和建模活动。该模块将通过圣何塞州立大学开发的基于网络的教育材料储存库分发给其他教员。

0853639RobertsIntellectual Merit - Non-native aggregation is a ubiquitous hurdle to successful over-expression and purification of recombinant proteins throughout the biotechnology and biopharmaceutical industries. This is particularly the case for multi-domain proteins, such as natural and designed antibodies, as well as for other predominantly-beta proteins. Fundamentally, rational design of these more complex proteins is handicapped by limitations in the mechanistic understanding of key features of sequence and structure that control aggregation of multi-domain proteins.This project seeks to lay a foundation both to fill the gaps in fundamental understanding of the mechanism(s) of multi-domain protein aggregation, and to provide first-generation design rules to imbue aggregation resistance. Human gamma-D Crystallin (gamma D-Crys) will be the model multidomain protein, chosen because it possesses a number of desirable features: (1) it is a single chain, two domain protein; (2) it represents an important set of all-beta proteins; (3) it has established aggregation and folding behavior; (4) it has available, aggregation-prone mutants. Broader Impact - The proposed research will result in an improved mechanistic understanding of aggregation and rational design of aggregation resistance for multi-domain, all-beta Greek-key proteins, of which a number of antibody constructs are a subclass of widespread biological and biotechnological interest. The research will also provide a framework for the education and training of graduate and undergraduate students in the PIs' laboratories, with a specific focus on cutting-edge experimental and modeling tools. As in the past, the PIs will involve students drawn from underrepresented groups in science and engineering. Finally, a set of examples and problems will be developed to incorporate aspects of this research into the undergraduate curricula, including computational and modeling activities. This module will be disseminated to other faculty via a web-based repository of educational materials developed at San Jose State University.