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

费尔南德斯智力优势-非天然聚集是生物技术和生物制药行业成功过度表达和纯化重组蛋白的普遍障碍。这尤其适用于多结构域蛋白质，如天然抗体和设计抗体，以及其他以β为主的蛋白质。从根本上说，这些更复杂的蛋白质的合理设计受到了对控制多域蛋白质聚集的序列和结构的关键特征的机械理解的限制。该项目旨在为填补对多域蛋白质聚集机制(S)的基本理解的空白，并提供第一代设计规则来灌输聚集阻力奠定基础。人类Gamma-D Crystallin(Gamma D-Crys)将是模型多结构域蛋白，因为它具有许多理想的特征：(1)它是一个单链，两个结构域的蛋白；(2)它代表一组重要的全β蛋白；(3)它已经建立了聚集和折叠行为；(4)它有可用的，容易聚集的突变体。更广泛的影响--拟议的研究将提高对聚集的机制的理解，并合理设计多结构域、全β希腊关键蛋白的聚集阻力，其中一些抗体结构是广泛的生物学和生物技术兴趣的子类。这项研究还将为PIS实验室的研究生和本科生的教育和培训提供一个框架，特别侧重于尖端的实验和建模工具。一如以往，督导计划将邀请来自理工科代表性较低组别的学生参加。最后，将开发一组例子和问题，以将这项研究的各个方面纳入本科课程，包括计算和建模活动。该单元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传播给其他教员。

0853543FernandezIntellectual 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.