Engineering Protein Stability Through Phage-Assisted Continuous Evolution

 描述(申请人提供)：蛋白质稳定性是异源表达、生物催化和治疗应用的重要要求。然而，稳定蛋白质突变的发现在蛋白质工程领域仍然是一个挑战。由于对序列与稳定性的关系缺乏了解，通过合理的设计来鉴定稳定突变仍然是困难的。另一方面，定向进化方法虽然需要很少的结构-活性关系的先验知识，但主要依赖于使用融合报告构建来选择稳定的蛋白质。这些方法受到蛋白质大小和融合报告干扰等因素的限制。此外，在许多情况下，只选择稳定性，这可能会导致意想不到的后果，如失去蛋白质活性。 这项拟议的研究试图开发一种通用的方法来快速进化稳定的蛋白质，同时确保通过噬菌体辅助的持续进化(PACE)保持蛋白质的活性。PACE允许在一周内进行数百轮定向进化，而只需最少的研究人员干预。通过将负选择与大肠杆菌热休克反应西格玛因子σ32的激活联系起来，所提出的方法将使用PACE来针对不稳定的蛋白质进行选择。蛋白质的功能可以通过同时对感兴趣的活动进行正选择来维持。在概念验证实验中验证了所提出的系统之后，它将被应用于进化的体内，能够结合和中和突变的亨廷顿蛋白错误折叠和细胞毒性。

 DESCRIPTION (provided by applicant): Protein stability is an important requirement for heterologous expression, biocatalysis, and therapeutic applications. However, the discovery of stabilizing protein mutations remains a challenge in the field of protein engineering. Identifying stabilizing mutations through rational design remains difficult due to the poor understanding of how sequence relates to stability. On the other hand, directed evolution methods, while requiring little prior knowledge of structure-activity relationships, have mainly relied on using fusion reporter constructs for selection of stable proteins. These approaches are limited by factors such as protein size and interference from the fused reporter. Additionally, in many cases only stability is selected for, which can lead to unintended consequences such as loss of protein activity. The proposed research seeks to develop a general method for rapidly evolving stable proteins while ensuring maintenance of protein activity through Phage-assisted continuous evolution (PACE). PACE allows hundreds of rounds of directed evolution to be performed within the space of a week with minimal researcher intervention. By linking negative selection to activation of the E. coli heat shock response sigma factor σ32, the proposed method will use PACE to select against destabilized proteins. Protein function can be maintained by simultaneously conducting positive selection for an activity of interest. After the proposed system is validated i proof-of-concept experiments, it will be applied to evolving intrabodies capable of binding to and neutralizing mutant huntingtin misfolding and cytotoxicity.