Biofoundry-assisted expression and characterization of plant proteins.
Biofoundry-assisted expression and characterization of plant proteins.
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植物蛋白的生物基础辅助表达和表征。
DOI：
10.1093/synbio/ysab029
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Patron NJ
中科院分区：
文献类型：
--
作者：
Dudley QM;Cai YM;Kallam K;Debreyne H;Carrasco Lopez JA;Patron NJ
合成生物学的许多目标，包括生物合成途径的阐明和重构以及调节电路和网络的工程，都需要蛋白质功能的知识。在植物中，大基因家族的盛行意味着将特定功能与个别蛋白质联系起来可能特别具有挑战性。然而，蛋白质的特性仍然是一个技术瓶颈，经常需要大量的努力来优化表达和纯化方案。为了利用生物芯片的能力来加快设计-构建-测试-学习周期，我们提出了一种自动化DNA组装和植物蛋白质无细胞表达的工作流程，该工作流程加快了优化并实现了酶活性的快速筛选。首先，我们开发了一个与植物砖兼容的金门DNA组装工具箱，其中包含用于使用Escherichiacoli或小麦胚芽裂解物进行无细胞表达的质粒受体，以及一组用于检测、纯化和改进表达/折叠的N-端和C-端标签部分。接下来，我们使用声学液体处理平台优化了小型化无细胞反应的自动组装，然后比较了标签配置，以确定哪些提高了表达。此外，我们还开发了一种基于荧光素酶的快速定量系统，该系统需要最少的11个氨基酸标签，并演示了合成后标签的轻松移除。最后，我们展示了几种功能分析可以在不需要蛋白质纯化的情况下进行无细胞蛋白质合成反应。总之，DNA部件的自动组装和无细胞表达反应的结合将显著增加测试和了解植物蛋白质功能的实验的吞吐量，并使DNA部件能够在下游植物工程工作流程中直接重复使用。
Many goals in synthetic biology, including the elucidation and refactoring of biosynthetic pathways and the engineering of regulatory circuits and networks, require knowledge of protein function. In plants, the prevalence of large gene families means it can be particularly challenging to link specific functions to individual proteins. However, protein characterization has remained a technical bottleneck, often requiring significant effort to optimize expression and purification protocols. To leverage the ability of biofoundries to accelerate design–built–test–learn cycles, we present a workflow for automated DNA assembly and cell-free expression of plant proteins that accelerates optimization and enables rapid screening of enzyme activity. First, we developed a phytobrick-compatible Golden Gate DNA assembly toolbox containing plasmid acceptors for cell-free expression using Escherichiacoli or wheat germ lysates as well as a set of N- and C-terminal tag parts for detection, purification and improved expression/folding. We next optimized automated assembly of miniaturized cell-free reactions using an acoustic liquid handling platform and then compared tag configurations to identify those that increase expression. We additionally developed a luciferase-based system for rapid quantification that requires a minimal 11–amino acid tag and demonstrate facile removal of tags following synthesis. Finally, we show that several functional assays can be performed with cell-free protein synthesis reactions without the need for protein purification. Together, the combination of automated assembly of DNA parts and cell-free expression reactions should significantly increase the throughput of experiments to test and understand plant protein function and enable the direct reuse of DNA parts in downstream plant engineering workflows.
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