Curated Optogenetic Publication Database

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Gigavalent display of proteins on monodisperse polyacrylamide hydrogels as a versatile modular platform for functional assays and protein engineering.

violet PhoCl in vitro Extracellular optogenetics
bioRxiv, 31 Oct 2021 DOI: 10.1101/2021.10.30.466587 Link to full text
Abstract: The robust modularity of biological components that are assembled into complex functional systems is central to synthetic biology. Here we apply modular “plug and play” design principles to a microscale solid phase protein display system that enables protein purification and functional assays for biotherapeutics. Specifically, we capture protein molecules from cell lysates on polyacrylamide hydrogel display beads (‘PHD beads’), made in microfluidic droplet generators. These monodisperse PHD beads are decorated with predefined amounts of anchors, methacrylate-PEG-benzylguanine (BG) and methacrylate-PEG-chloroalkane (CA). Anchors form covalent bonds with fusion proteins bearing cognate tag recognition (SNAP and Halo-tags) in specific, orthogonal and stable fashion. Given that these anchors are copolymerised throughout the 3D structure of the beads, proteins are also distributed across the entire bead sphere, allowing attachment of ∼109 protein molecules per bead (Ø 20 μm). This mode of attachment reaches a higher density than possible on widely used surface-modified beads, and additionally mitigates surface effects that often complicate studies with proteins on beads. We showcase a diverse array of protein modules that enable the secondary capture of proteins, either non-covalently (IgG and SUMO-tag) or covalently (SpyCatcher, SpyTag, SnpCatcher and SnpTag). Proteins can be displayed in their monomeric forms, but also reformatted as a multivalent display (using secondary capture modules that create branches) to test the contributions of avidity and multivalency towards protein function. Finally, controlled release of modules by irradiation of light is achieved by incorporating the photocleavable protein PhoCl: irradiation severs the displayed protein from the solid support, so that functional assays can be carried out in solution. As a demonstration of the utility of valency engineering, an antibody drug screen is performed, in which an anti-TRAIL-R1 scFv protein is released into solution as monomers-hexamers, showing a ∼50-fold enhanced potency in the pentavalent format. The ease of protein purification on solid support, quantitative control over presentation and release of proteins and choice of valency make this experimental format a versatile, modular platform for large scale functional analysis of proteins, in bioassays of protein-protein interactions, enzymatic catalysis and bacteriolysis.
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