无细胞蛋白质合成：从基础研究到工程应用

doi:10.12211/2096-8280.2021-064

合成生物学 ›› 2022, Vol. 3 ›› Issue (3): 465-486.DOI: 10.12211/2096-8280.2021-064

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.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

收稿日期:2021-06-06 修回日期:2021-09-21 出版日期:2022-06-30 发布日期:2022-07-13
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

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

Jiaqi HOU^1,², Nan JIANG^1,², Lianju MA², Yuan LU¹

^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:2022-06-30 Published:2022-07-13
Contact: Lianju MA,Yuan LU

摘要/Abstract

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

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

中图分类号:

Q816

后佳琦, 姜楠, 马莲菊, 卢元. 无细胞蛋白质合成：从基础研究到工程应用[J]. 合成生物学, 2022, 3(3): 465-486.

Jiaqi HOU, Nan JIANG, Lianju MA, Yuan LU. Cell-free protein synthesis: from basic research to engineering applications[J]. Synthetic Biology Journal, 2022, 3(3): 465-486.

图/表 8

图1 每年CFPS论文发表数量

Fig. 1 Numbers of annual CFPS publications

图2 体内细胞和体外无细胞蛋白质合成比较（在体内细胞系统，目的质粒导入细胞，通过培养、裂解和离心纯化获得目的蛋白。在CFPS系统中，通过向细胞提取物中添加DNA模板、氨基酸、NTPs、能量底物等辅助因子，离心纯化后获得目的蛋白）

Fig. 2 Comparison of protein synthesis through in vivo cellular and in vitro cell-free systems(In the in vivo cellular system, the target plasmid is introduced into the cell, and the target protein is obtained through cell culture, lysis, and product purification. In the CFPS system, by adding DNA template, amino acids, NTPs, energy substrates and other cofactors to the cell extract, the target protein is obtained after centrifugation and purification.)

图3 无细胞蛋白质合成过程（无细胞体系的成分包括细胞提取物、辅助因子、DNA模板等，通过细胞提取物中的转录翻译机器，无细胞体系在一定条件下孵育几个小时即可产生目的蛋白）

Fig.3 Cell-free protein synthesis process(The components of the cell-free system include cell extracts, cofactors, DNA templates, etc. Through the transcription and translation machinery in the cell extracts, the cell-free system can produce the target protein after incubating for several hours under designated conditions.)

表1 不同提取物体系优缺点对比

Tab. 1 Comparison of advantages and disadvantages of different extract systems

细胞提取物类型	优点	缺点
大肠杆菌细胞	生长速度快、易于培养、产量高、成本效益高	在翻译后修饰、膜蛋白合成和其他难以合成的蛋白质合成方面存在局限性
酿酒酵母细胞	易于培养、可用于研究真核生物的转录翻译机制	蛋白质产量低，未显示充分的翻译后修饰
小麦胚芽细胞	蛋白质可溶性增强、可表达毒性蛋白质、产量高	需要去除各种抑制酶，操作流程较繁琐，合成复杂蛋白、进行翻译后修饰具有局限性
烟草细胞	有利于合成翻译后修饰蛋白质，还有助于糖基化和二硫键形成，相对其他真核平台耗时短	蛋白质产量一般，未显示充分的翻译后修饰
兔网织红细胞	具有翻译后修饰功能，可表达膜蛋白、毒性蛋白	蛋白质产量低，耗时长
中国仓鼠卵巢细胞	具有翻译后修饰功能，可表达膜蛋白、毒性蛋白	蛋白质产量较低
昆虫细胞	具有翻译后修饰功能，可表达膜蛋白、冰结构蛋白	蛋白质产量较低，无细胞体系中需要更多的提取物
PURE	组分纯化后无核酸酶或蛋白酶残留，灵活和模块化	成本较高，不能激活内源性代谢

图4 CFPS反应模式（a）批式模式。所有反应成分添加到一个管中进行，操作简单、便捷。（b）连续交换。通过半透膜，营养物质和产生的代谢副产物分隔开，克服了代谢产物对体系的抑制作用。（c）套管。气体通过气泵不断地泵入到内管中，显著提高氧的传递效率，提高产量。（d）数字微流控。数字微流控技术可控制单个液滴的移动、混合、分离等，具有良好的交互性和灵活性

Fig. 4 Modes for CFPS operation(a) Batch mode. All reaction components are added to one tube, which is simple and convenient for operating. (b) Continuous exchange. Through the semi-permeable membrane, nutrients are separated from the metabolic by-products, which overcomes the inhibitory effect of the metabolic products on the reaction. (c) Tube in tube. The gas is continuously pumped into the inner tube through the air pump, which significantly improves the efficiency of oxygen transfer and increases the output. (d) Digital microfluidic technology. It can control the movement, mixing, and separation of a single droplet, with good interactivity and flexibility.

图5 基因电路原型设计（转录单位由单独质粒或线性DNA模板进行编码，可以作为逻辑门的类似物，然后通过无细胞基因表达反应在体外执行分子计算或遗传程序，以预测电路在电路在细胞中的功能）

Fig. 5 Prototype designs for genetic circuits(The transcription unit is encoded by a single plasmid or linear DNA template, which can be used as an analog of a logic gate, and then molecular calculations or genetic programs are performed in vitro through a cell-free gene expression reaction to predict the function of the circuit in the cell.)

图6 无细胞蛋白质工程的应用（无细胞蛋白质工程的应用主要包括：膜蛋白、类病毒颗粒、翻译后修饰、非天然氨基酸的嵌入和蛋白质进化）

Fig. 6 Applications of cell-free protein engineering(The applications of cell-free protein engineering mainly include membrane proteins, virus-like particles, post-translational modification, unnatural amino acid intercalation, and protein evolution)

图7 人工细胞的构建（分子拥挤、区室化、基因噪声、网络、动态行为和细胞通讯是维持一个细胞正常生命活动所必须的结构和功能特征，能有效调控操作细胞的正常运行）

Fig. 7 Construction of artificial cells(Molecular crowding, compartmentalization, gene noise, networks, dynamic behaviors and cell communications are the structural and functional characteristics necessary to maintain the normal life activities of a cell, which can effectively regulate and operate the cell)

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无细胞蛋白质合成：从基础研究到工程应用

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

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