Abstract
Monobodies are synthetic non-immunoglobulin customizable protein binders invaluable to basic and applied research, and of considerable potential as future therapeutics and diagnostic tools. The ability to reversibly control their binding activity to their targets on demand would significantly expand their applications in biotechnology, medicine, and research. Here we present, as proof-of-principle, the development of a light-controlled monobody (OptoMB) that works in vitro and in cells and whose affinity for its SH2-domain target exhibits a 330-fold shift in binding affinity upon illumination. We demonstrate that our αSH2-OptoMB can be used to purify SH2-tagged proteins directly from crude E. coli extract, achieving 99.8% purity and over 40% yield in a single purification step. By virtue of their ability to be designed to bind any protein of interest, OptoMBs have the potential to find new powerful applications as light-switchable binders of untagged proteins with the temporal and spatial precision afforded by light.
Highlights
Monobodies are synthetic non-immunoglobulin customizable protein binders invaluable to basic and applied research, and of considerable potential as future therapeutics and diagnostic tools
Our strategy to develop a light-sensitive HA4 was to design various chimeras of this monobody with AsLOV2 from A. sativa, and test their ability to bind and release the SH2 domain depending on light conditions
Given the large conformational change of AsLOV2 triggered by light (Fig. 1a), our hypothesis was that the native conformation of the monobody domain in some chimeras would be preserved in the dark, allowing it to bind to SH2, but disrupted in the light, causing it to release its target
Summary
Monobodies are synthetic non-immunoglobulin customizable protein binders invaluable to basic and applied research, and of considerable potential as future therapeutics and diagnostic tools. 1234567890():,; Monobodies[1] and other synthetic non-immunoglobulin protein-binding scaffolds, such as affibodies, anticalins, and DARPins, bind to their targets with affinities and selectivities typically found in antibodies[2,3,4,5], yet they are much simpler in structure Because of their ability to be designed to bind a variety of proteins of interest, monobodies have become invaluable tools for biomedical research and biotechnology[6,7,8,9]. Monobodies are synthetic proteins derived from the 10th domain of human fibronectin type III While they were originally designed to functionally resemble nanobodies[1,6,13], monobodies feature unique structural advantages such as a reduced size (20–25% smaller) and a compact protein core without disulfide bridges[1,7]. The Jα helix is packed against the core of the protein[19,20]
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