The assembly of robust, modular biological components into complex functional systems is central to synthetic biology. Here, we apply modular “plug and play” design principles to a solid-phase protein display system that facilitates protein purification and functional assays. Specifically, we capture proteins 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), that react covalently with SNAP-/Halo-tag fusion proteins, respectively, in a specific, orthogonal, and stable fashion. Anchors, and thus proteins, are distributed throughout the entire bead volume, allowing attachment of ∼10⁹ protein molecules per bead (⌀ 20 μm) ─a higher density than achievable with commercial surface-modified beads. We showcase a diverse array of protein modules that enable the secondary capture of proteins, either noncovalently (IgG and SUMO-tag) or covalently (SpyCatcher, SpyTag, SnpCatcher, and SnpTag), in mono- and multivalent display formats. Solid-phase protein binding and enzymatic assays are carried out, and incorporating the photocleavable protein PhoCl enables the controlled release of modules via visible-light irradiation for functional assays in solution. We utilize photocleavage for valency engineering of an anti-TRAIL-R1 scFv, enhancing its apoptosis-inducing potency ∼50-fold through pentamerization.

中文翻译：

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单分散聚丙烯酰胺水凝胶上蛋白质的千兆价展示作为功能测定和蛋白质工程的多功能模块化平台

将强大的模块化生物组件组装成复杂的功能系统是合成生物学的核心。在这里，我们将模块化“即插即用”设计原则应用于促进蛋白质纯化和功能测定的固相蛋白质展示系统。具体来说，我们在由微流体液滴发生器制成的聚丙烯酰胺水凝胶展示珠（PHD 珠）上捕获蛋白质。这些单分散的 PHD 珠子装饰有预定义数量的锚，甲基丙烯酸酯-PEG-苄基鸟嘌呤 (BG) 和甲基丙烯酸酯-PEG-氯代烷 (CA)，它们分别与 SNAP-/Halo-tag 融合蛋白以特定的正交方式发生共价反应，和稳定的时尚。锚点和蛋白质分布在整个珠子体积中，允许附着 ∼10 ⁹每个珠子的蛋白质分子 (⌀ 20 μm) ─ 比商业表面改性珠子的密度更高。我们展示了多种蛋白质模块，它们能够以单价和多价展示格式对蛋白质进行二次捕获，无论是非共价（IgG 和 SUMO-tag）还是共价（SpyCatcher、SpyTag、SnpCatcher 和 SnpTag）。进行固相蛋白质结合和酶促测定，并结合光可裂解蛋白质 PhoCl 可以通过可见光照射控制模块的释放，用于溶液中的功能测定。我们利用光裂解对抗 TRAIL-R1 scFv 进行化合价工程，通过五聚体化将其诱导细胞凋亡的效力提高 50 倍。