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 ?32, 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.
Engineered proteins are important for both research and therapeutic applications, but protein instability has presented a persistent and difficult challeng. The proposed research aims to develop a general method for improving protein stability through directed evolution. Additionally, the proposed method will be applied toward evolving intrabodies capable of rehabilitating the mutant huntingtin protein responsible for Huntington's Disease.
| Wang, Tina; Badran, Ahmed H; Huang, Tony P et al. (2018) Continuous directed evolution of proteins with improved soluble expression. Nat Chem Biol 14:972-980 |
