Synthetic Biology Journal ›› 2022, Vol. 3 ›› Issue (3): 465-486.DOI: 10.12211/2096-8280.2021-064

• Invited Review • Previous Articles     Next Articles

Cell-free protein synthesis: from basic research to engineering applications

Jiaqi HOU1,2, Nan JIANG1,2, Lianju MA2, Yuan LU1   

  1. 1.Department of Chemical Engineering,Tsinghua University,Beijing 100084,China
    2.College of Life Sciences,Shenyang Normal University,Shenyang 110034,Liaoning,China
  • Received:2021-06-06 Revised:2021-09-21 Online:2022-07-13 Published:2022-06-30
  • Contact: Lianju MA, Yuan LU

无细胞蛋白质合成:从基础研究到工程应用

后佳琦1,2, 姜楠1,2, 马莲菊2, 卢元1   

  1. 1.清华大学化学工程系,北京 100084
    2.沈阳师范大学生命科学学院,辽宁 沈阳 110034
  • 通讯作者: 马莲菊,卢元
  • 作者简介:后佳琦(1994—),女,硕士研究生。研究方向为无细胞生物合成。 E-mail:2580070932@qq.com
    姜楠(1995—),女,硕士研究生。研究方向为无细胞生物合成。 E-mail:1023796982@qq.com
    马莲菊(1969—),女,教授,硕士生导师。研究方向为资源与应用微生物学等。 E-mail:malianju@163.com
    卢元(1983—),男,副教授,博士生导师。研究方向为合成生物学、生物大分子工程等。 E-mail:yuanlu@tsinghua.edu.cn
  • 基金资助:
    国家自然科学基金(21878173);北京市自然科学基金(2192023);国家重点研发计划(2018YFA0901700);清华大学实验室创新基金

Abstract:

Cell-free protein synthesis (CFPS), also known as in vitro gene expression, is a multifunctional technique used to complement cell-based protein expression, which is at the core of cell-free synthetic biology. Since the CFPS system does not require a living cell, it can simulate the entire cellular transcription and translation process in vitro in a controlled environment, and allows for an in-depth study of individual components and biological networks. Therefore, as a platform technology, it is expected to overcome the loopholes caused by the limitations of cell membranes in the current in vivo manufacturing systems, which has a broad research prospect in fundamental and applied scientific research. The cell-free operation is simple and easy to control, and its advantages over in vivo protein expression include its nature with open systems, eliminating the dependence on living cells and using all system energy for the production of the target proteins. This article reviews the composition of CFPS systems and their development based on different component types, including different biological extracts or purified transcription and translation components. Furthermore, different CFPS reaction patterns are introduced, including batch and continuous exchange modes, and the research progress of CFPS systems in genetic circuits, protein engineering, and the construction of artificial life is described. Among them, the genetic circuit research progress mainly summarizes the latest applications and contributions of cell-free technology in the prototype design, biosensors, and in vitro metabolic engineering. The protein engineering research progress lists the advantages and advances of the CFPS systems for producing membrane proteins, virus-like particles, post-translational modifications, unnatural amino acid incorporation and protein evolution. In the construction of artificial "living systems", the synthesis of bacteriophages and the construction of artificial cells have opened up a novel frontier field. Finally, the opportunities and challenges of the CFPS platforms for future scientific research and industrial applications are highlighted.

Key words: cell-free protein synthesis, genetic circuits, protein engineering, artificial cells

摘要:

无细胞蛋白质合成是无细胞合成生物学的技术核心,亦被称为体外蛋白质翻译,是一种用于补充基于细胞的蛋白质表达的技术。无细胞蛋白质合成系统无需完整的活细胞就可以在体外受控环境中模拟整个细胞的转录和翻译过程,并允许对单个成分和反应网络进行详细深入的研究。因此,无细胞蛋白质合成作为一种平台技术,有望克服当前胞内生产系统中因为细胞膜约束带来的表达局限性,在基础科学研究和应用科学研究中具有广阔的前景。无细胞系统操作简单、便于控制,相对于体内蛋白质表达,其优势还包括其开放特性、消除对活细胞的依赖以及将所有系统物质能量集中在目标蛋白质生产上。本文首先概述了无细胞蛋白质合成系统的组成及基于不同组件类型的无细胞蛋白质合成系统的发展,包括以不同生物提取物为基础的系统以及使用重组元素的蛋白质合成体系。之后介绍了以分批反应、连续交换为代表的无细胞蛋白质合成系统的不同反应模式,阐述了无细胞在基因电路、蛋白质工程和人工“生命体系”构建中的应用和研究进展。其中,基因电路主要概述了无细胞技术在原型设计、生物传感、代谢工程三个方面的最新应用;蛋白质工程依次罗列了无细胞技术在膜蛋白、类病毒颗粒、翻译后修饰、非天然氨基酸嵌入以及蛋白质进化等方面的应用拓展;在人工“生命体系”构建中,噬菌体的合成和人工细胞的构筑开辟了新的前沿领域。最后文章分析了无细胞蛋白质合成系统在未来进一步的科学研究和工业化应用中面临的机遇和挑战。

关键词: 无细胞蛋白质合成, 基因电路, 蛋白质工程, 人工细胞

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