基于水凝胶颗粒的蛋白质显示通过颗粒模板化乳化†实现

IF 3.9 3区 化学 Q2 CHEMISTRY, MULTIDISCIPLINARY
RSC Advances Pub Date : 2025-07-23 DOI:10.1039/D5RA03622D
Han Wu, Jiayao Fang, Jiao Chen, Yaoqi Wang and Bo Zheng
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引用次数: 0

摘要

蛋白质展示技术实现了高通量筛选,在蛋白质发现和工程中发挥着重要作用。传统的体内展示方法面临着基因转化效率低下和细胞增殖动力学复杂等挑战,而体外展示方法往往局限于基于亲和力的选择,并且由于反应条件同质而存在表达偏差。在这里,我们提出了一种基于水凝胶颗粒的蛋白质展示方法,该方法通过颗粒模板乳化实现。该方法使用功能化聚丙烯酰胺水凝胶颗粒作为分离的微反应器,结合DNA引物用于基因型固定和Ni-NTA组用于捕获组氨酸标记的蛋白质表型。所展示的蛋白质是通过在分离的液滴内无细胞蛋白表达合成的,克服了体内细胞培养的局限性,实现了区隔筛选,这在传统的均质体外系统中是具有挑战性的。使用颗粒模板乳化,单个水凝胶颗粒可以在30秒内与单个DNA模板快速封装成分离的油包水滴,而无需专门的仪器。多达109个颗粒可以乳化在一个标准的50毫升锥形管。与传统的液滴微流体相比,颗粒模板乳化实现了更高的单颗粒封装,提高了一对一颗粒- DNA配对效率,减少了试剂消耗,最大限度地减少了因配对不当造成的DNA文库损失。在液滴内依次进行数字PCR和无细胞蛋白表达,将扩增的DNA和表达的蛋白固定在同一颗粒上,从而建立稳定的基因型-表型连锁。该方法消除了细胞处理的需要,实现了分区功能筛选,并为蛋白质展示提供了快速,可扩展和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。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Hydrogel particle-based protein display enabled by particle-templated emulsification†

Hydrogel particle-based protein display enabled by particle-templated emulsification†

Protein display technology enables high-throughput screening and plays an important role in protein discovery and engineering. Conventional in vivo display methods face challenges such as inefficient gene transformation and complex cell proliferation dynamics, while in vitro 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 via cell-free protein expression within isolated droplets, overcoming the limitations of in vivo cell culture and enabling compartmentalized screening, which is challenging in conventional homogeneous in vitro 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 109 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.

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来源期刊
RSC Advances
RSC Advances chemical sciences-
CiteScore
7.50
自引率
2.60%
发文量
3116
审稿时长
1.6 months
期刊介绍: An international, peer-reviewed journal covering all of the chemical sciences, including multidisciplinary and emerging areas. RSC Advances is a gold open access journal allowing researchers free access to research articles, and offering an affordable open access publishing option for authors around the world.
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