Engineering Scaffolds for Modular Proteins Assembly

摘要

Proteins associate with each other to form complex collaborative network in order to perform certain functions, such as multi-step reactions in metabolic process, receiving and transferring signals in signaling pathways, and host cell infection. Scaffolds have evolved in nature to organize proteins to enhance collaborative efficiency. Inspired by features and advantages of natural scaffolds, synthetic scaffolds are developed for customized applications by organizing proteins into functional modules.;In the first objective of this thesis, DNA was used as a scaffold to organize 5 proteins involved in cellulosic fuel cell. DNA scaffolds feature simple hybridization rules and precise programming of structures. These features allow precise control of order and distance of the 5 proteins, enabling investigation on substrate channeling effect of adjacent enzymes and synergistic effect of enzymes in close proximity. Although DNA can be programmed into structures in 1 to 3-dimenions, the heavy programming load and cost prevented the use of DNA as 3-dimensional scaffolds, leading to the exploration of native 3-dimensional scaffolds.;In the second objective, functionalization of protein nanoparticles was investigated to develop 3-dimensional scaffolds, which plays an important role in signal amplification of biosensors, drug delivery and biocatalysis. Native protein nanoparticles can self-assemble into stable and biocompatible structures. Previous functionalization methods can lead to incorrect folding of 3-dimensional structures or loss of control over specific modifications that limit and complicate the application of protein nanoparticles. To develop a simple and modular functionalization method, an enzyme-mediated ligation strategy was investigated; sortase-mediated ligation enabled specific conjugation of functional proteins onto E2 protein nanoparticles from Bacillus stearothermophilus, a model 3-dimesnional scaffold.;Finally, to further simplify the use of native 3-dimensional scaffolds, one-pot synthesis of protein scaffolds that are ready to use after production were developed. It is inspired by the biogenesis of outer membrane vesicles (OMVs), which naturally form into proteoliposomes during growth cycle of bacteria. They feature the simultaneous formation of nanostructures and displaying of functional proteins. Membrane proteins were investigated as anchors for displaying heterologous proteins, which allowed simultaneous display of proteins of different functionalities, such as detection and reporter moieties.