Wu Han, Fang Jiayao, Chen Jiao, Wang Yaoqi, Zheng Bo
Institute of Chemical Biology, Shenzhen Bay Laboratory Shenzhen China
RSC Adv. 2025 Jul 23;15(32):26362-26370. doi: 10.1039/d5ra03622d. eCollection 2025 Jul 21.
Protein display technology enables high-throughput screening and plays an important role in protein discovery and engineering. Conventional display methods face challenges such as inefficient gene transformation and complex cell proliferation dynamics, while display methods are often limited to affinity-based selection and suffer from expression bias due to homogeneous reaction conditions. Here, we present a hydrogel particle-based protein display method enabled by particle-templated emulsification. This approach uses functionalized polyacrylamide hydrogel particles as isolated microreactors, incorporating DNA primers for genotype immobilization and Ni-NTA groups for capturing histidine-tagged protein phenotypes. Displayed proteins are synthesized cell-free protein expression within isolated droplets, overcoming the limitations of cell culture and enabling compartmentalized screening, which is challenging in conventional homogeneous systems. Using particle-templated emulsification, single hydrogel particles can be rapidly encapsulated with individual DNA templates into isolated water-in-oil droplets within 30 seconds, without the need for specialized instrumentation. Up to 10 particles can be emulsified in a standard 50 ml conical tube. Compared to conventional droplet microfluidics, particle-templated emulsification achieves higher single-particle encapsulation and improved one-to-one particle-DNA pairing efficiency, reducing reagent consumption and minimizing DNA library loss caused by improper pairing. Digital PCR and cell-free protein expression are sequentially performed within droplets, with both the amplified DNA and the expressed protein immobilized on the same particle, thereby establishing a stable genotype-phenotype linkage. This method eliminates the need for cell handling, enables compartmentalized functional screening, and provides a fast, scalable, and user-friendly workflow for protein display, offering strong potential in directed evolution and protein engineering.
蛋白质展示技术能够实现高通量筛选,在蛋白质发现和工程中发挥着重要作用。传统的展示方法面临着诸如基因转化效率低下和细胞增殖动力学复杂等挑战,而展示方法通常局限于基于亲和力的选择,并且由于均相反应条件而存在表达偏差。在此,我们提出一种基于水凝胶颗粒的蛋白质展示方法,该方法由颗粒模板乳化实现。这种方法使用功能化的聚丙烯酰胺水凝胶颗粒作为孤立的微反应器,并入用于固定基因型的DNA引物和用于捕获组氨酸标签蛋白表型的Ni-NTA基团。展示的蛋白质在孤立的液滴内通过无细胞蛋白质表达合成,克服了细胞培养的局限性并实现了分区筛选,这在传统的均相系统中具有挑战性。使用颗粒模板乳化,单个水凝胶颗粒可在30秒内用单个DNA模板快速封装到孤立的油包水液滴中,无需专门的仪器。在标准的50毫升锥形管中最多可乳化10个颗粒。与传统的液滴微流控相比,颗粒模板乳化实现了更高的单颗粒封装和提高的一对一颗粒-DNA配对效率,减少了试剂消耗并最大限度地减少了因配对不当导致的DNA文库损失。数字PCR和无细胞蛋白质表达在液滴内依次进行,扩增的DNA和表达的蛋白质都固定在同一颗粒上,从而建立稳定的基因型-表型联系。这种方法无需细胞处理,实现了分区功能筛选,并为蛋白质展示提供了一种快速、可扩展且用户友好的工作流程,在定向进化和蛋白质工程中具有强大的潜力。
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