Fryer Thomas, Rogers Joel David, Mellor Christopher, Kohler Timo N, Minter Ralph, Hollfelder Florian
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom.
Antibody Discovery and Protein Engineering, R&D, AstraZeneca, Milstein Building, Granta Park, Cambridge CB21 6GH, United Kingdom.
ACS Cent Sci. 2022 Aug 24;8(8):1182-1195. doi: 10.1021/acscentsci.2c00576. Epub 2022 Aug 1.
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 ∼10 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.
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。在此，我们将模块化的“即插即用”设计原则应用于一个有助于蛋白质纯化和功能分析的固相蛋白质展示系统。具体而言，我们将蛋白质捕获在微流控液滴发生器中制备的聚丙烯酰胺水凝胶展示珠（PHD珠）上。这些单分散的PHD珠用预定义量的锚定物修饰，即甲基丙烯酸酯-聚乙二醇-苄基鸟嘌呤（BG）和甲基丙烯酸酯-聚乙二醇-氯代烷烃（CA），它们分别以特定、正交且稳定的方式与SNAP-/Halo标签融合蛋白发生共价反应。锚定物以及蛋白质分布在整个珠子体积中，使得每个珠子（直径20μm）能够附着约10个蛋白质分子——这一密度高于商业表面修饰珠子所能达到的密度。我们展示了一系列多样的蛋白质模块，这些模块能够以单价和多价展示形式，通过非共价（IgG和SUMO标签）或共价（SpyCatcher、SpyTag、SnpCatcher和SnpTag）方式实现蛋白质的二次捕获。我们进行了固相蛋白质结合和酶促分析，并且引入可光裂解的蛋白质PhoCl能够通过可见光照射实现模块的可控释放，以便在溶液中进行功能分析。我们利用光裂解进行抗TRAIL-R1单链抗体片段（scFv）的价态工程，通过五聚化将其诱导凋亡的效力提高了约50倍。
