Improved Methods for the Development and Adaptation of Protein Switches
Nicholes, Nathan D
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Protein switches are protein fusions in which the activity of one protein is influenced by a signal from the other protein. Protein switches have a variety of potential applications as biosensors, therapeutics and research tools. At present, creating a new protein switch requires a time- and labor-intensive process of library creation, selection and characterization. This dissertation discusses how successful switch topologies can be used to develop new switches and efforts to develop modular protein switches. Protein switches created prior to this work using random insertions of β-lactamase (BLA) into ribose-binding protein and glucose-binding protein from E. coli were used to predict BLA insertion sites into xylose-binding protein (XBP). The creation of a xylose-responsive BLA fusion required the variation of the linkers connecting XBP and BLA as well as a change in the circular permutation of BLA indicating that insertion sites from previous switches may serve as a starting point for switch creation in the development of new protein switches. The feasibility of a modular protein switch was also studied using two different antibody mimetic scaffolds: DARPins and monobodies. A modular protein switch involves a protein fusion with a variable recognition domain. The objective of this effort was to use library creation techniques to identify a topology with the capability of maintaining the switching activity of the fusion after the binding interface is modified to recognize a different target ligand. Libraries of protein fusions were created by inserting BLA throughout the two scaffolds with different amino acid linkers between the two proteins. Fusions were identified that showed increased BLA activity in the presence of the cognate ligand (maltose binding protein from E. coli). The binding surfaces of the fusions with the largest, ligand-induced difference in BLA activity were then modified to recognize new ligands without otherwise altering the protein fusion. The monobody-BLA and DARPin-BLA fusions show promise as in vivo and in vitro protein switch scaffolds, however, efforts are needed to improve their specificity and stability. Advances in the development of protein switches using semi-rational design and antibody mimetics foreshadow more rapid exploration of both new switches and applications.