生物技术通报 ›› 2022, Vol. 38 ›› Issue (12): 100-114.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0346
收稿日期:
2022-03-23
出版日期:
2022-12-26
发布日期:
2022-12-29
作者简介:
许博楠,女,硕士研究生,研究方向:合成生物学;E-mail:基金资助:
XU Bo-nan(), FENG Jia, ZHOU Jian-ting, JIANG Jian-lan()
Received:
2022-03-23
Published:
2022-12-26
Online:
2022-12-29
摘要:
无细胞蛋白合成系统(cell-free protein synthesis,CFPS)是一种体外蛋白质合成的高效平台,与体内蛋白合成系统相比,它突破了活细胞的限制,使蛋白合成反应可以在试管中进行,具有灵活、高效、可控性强的特点。近几年来,合成生物学领域新技术的涌现推动了CFPS系统的快速发展,尤其在细胞提取物和模板DNA方面。细胞提取物和模板DNA是CFPS系统中不可或缺的重要组成,很大程度上决定了无细胞反应的效率和成本。本文综述了CFPS系统中细胞提取物和模板DNA方面的研究进展,主要介绍了最近新开发的细胞提取物系统的特点和应用前景,以及质粒DNA模板和线性DNA模板的研究进展。
许博楠, 冯佳, 周见庭, 蒋建兰. 无细胞蛋白合成系统中细胞提取物和模板DNA的研究进展[J]. 生物技术通报, 2022, 38(12): 100-114.
XU Bo-nan, FENG Jia, ZHOU Jian-ting, JIANG Jian-lan. Research Progress of Cell Extracts and Template DNAs in Cell-free Protein Synthesis System[J]. Biotechnology Bulletin, 2022, 38(12): 100-114.
细胞提取物系统 System | 优势 Advantages | 劣势 Disadvantages | 应用 Applications |
---|---|---|---|
真核细胞 Eukaryotic | |||
小麦胚芽 Wheat germ extract | 无需密码子优化;可高通量合成复杂蛋白质;可合成二硫键桥连的蛋白质;可实现多种蛋白质的正确折叠,使蛋白质具有高溶解性 | 细胞提取物制备费时且成本高;有限的翻译后修饰;缺乏内源性膜结构 | 蛋白质微阵列技术[ |
兔网织红细胞 Rabbit reticulocyte extract | 完善且成熟的体系;哺乳动物系统 | 提取物制备需处理活体动物;翻译效率低;需要添加外源性微粒体使蛋白质折叠 | 蛋白质微阵列技术[ |
昆虫细胞 Insect cell extract | 细胞易破碎;可进行翻译后修饰;具有内源性微粒体 | 细胞培养成本高;遗传操作少 | 膜蛋白的自动化生产[ |
仓鼠卵巢细胞 CHO cell extract | 可进行翻译后修饰;具有内源性微粒体 | 细胞培养成本高 | 生产糖基化蛋白[ |
酵母 Yeast extract | 细胞培养快速,提取物制备简单;遗传操作成熟;实现蛋白质的正确折叠;可进行糖基化修饰 | 无类似哺乳动物系统的翻译后修饰;蛋白产量低 | 生产病毒样颗粒用于抗病毒药物研究[ |
烟草细胞 Tobacco extract | 细胞提取物制备快速;可进行糖基化修饰和形成二硫键 | 含有内源氨基酸,会影响蛋白质产量 | 研究植物RNA病毒翻译复制机制[ |
人类细胞系 Human cell lines | 可进行翻译后修饰;具有内源性微粒体 | 细胞培养成本高;蛋白产量低 | 研究病毒复制机制[ |
原核细胞 Prokaryotic | |||
大肠杆菌 E. coli extract | 细胞提取物制备简单,成本低;蛋白产量高;遗传操作简便;应用范围广 | 缺乏具有翻译活性的内源性微粒体,有限的翻译后修饰;无法正确折叠复杂蛋白质 | 蛋白的高通量表达[ |
枯草芽孢杆菌 B. subtilis extract | 无需密码子优化;遗传操作成熟 | 蛋白质产量低;无翻译后修饰;无内源性微粒体 | 启动子库的原型构建[ |
链霉菌 Streptomyces extract | 细胞提取物制备简单 | 无翻译后修饰(未见报道);无内源性微粒体 | 表达高GC含量基因[ |
恶臭假单胞菌 P. putida extract | 细胞提取物制备简单 | 目前为止应用有限 | 筛选基因调控元件[ |
需钠弧菌 V. natriegens extract | 无需密码子优化;可产生大量有活性的提取物;每个细胞的核糖体浓度高 | 目前为止应用有限 | 启动子库的原型构建[ |
表1 不同细胞提取物系统的特点
Table 1 Features of different cell extract systems
细胞提取物系统 System | 优势 Advantages | 劣势 Disadvantages | 应用 Applications |
---|---|---|---|
真核细胞 Eukaryotic | |||
小麦胚芽 Wheat germ extract | 无需密码子优化;可高通量合成复杂蛋白质;可合成二硫键桥连的蛋白质;可实现多种蛋白质的正确折叠,使蛋白质具有高溶解性 | 细胞提取物制备费时且成本高;有限的翻译后修饰;缺乏内源性膜结构 | 蛋白质微阵列技术[ |
兔网织红细胞 Rabbit reticulocyte extract | 完善且成熟的体系;哺乳动物系统 | 提取物制备需处理活体动物;翻译效率低;需要添加外源性微粒体使蛋白质折叠 | 蛋白质微阵列技术[ |
昆虫细胞 Insect cell extract | 细胞易破碎;可进行翻译后修饰;具有内源性微粒体 | 细胞培养成本高;遗传操作少 | 膜蛋白的自动化生产[ |
仓鼠卵巢细胞 CHO cell extract | 可进行翻译后修饰;具有内源性微粒体 | 细胞培养成本高 | 生产糖基化蛋白[ |
酵母 Yeast extract | 细胞培养快速,提取物制备简单;遗传操作成熟;实现蛋白质的正确折叠;可进行糖基化修饰 | 无类似哺乳动物系统的翻译后修饰;蛋白产量低 | 生产病毒样颗粒用于抗病毒药物研究[ |
烟草细胞 Tobacco extract | 细胞提取物制备快速;可进行糖基化修饰和形成二硫键 | 含有内源氨基酸,会影响蛋白质产量 | 研究植物RNA病毒翻译复制机制[ |
人类细胞系 Human cell lines | 可进行翻译后修饰;具有内源性微粒体 | 细胞培养成本高;蛋白产量低 | 研究病毒复制机制[ |
原核细胞 Prokaryotic | |||
大肠杆菌 E. coli extract | 细胞提取物制备简单,成本低;蛋白产量高;遗传操作简便;应用范围广 | 缺乏具有翻译活性的内源性微粒体,有限的翻译后修饰;无法正确折叠复杂蛋白质 | 蛋白的高通量表达[ |
枯草芽孢杆菌 B. subtilis extract | 无需密码子优化;遗传操作成熟 | 蛋白质产量低;无翻译后修饰;无内源性微粒体 | 启动子库的原型构建[ |
链霉菌 Streptomyces extract | 细胞提取物制备简单 | 无翻译后修饰(未见报道);无内源性微粒体 | 表达高GC含量基因[ |
恶臭假单胞菌 P. putida extract | 细胞提取物制备简单 | 目前为止应用有限 | 筛选基因调控元件[ |
需钠弧菌 V. natriegens extract | 无需密码子优化;可产生大量有活性的提取物;每个细胞的核糖体浓度高 | 目前为止应用有限 | 启动子库的原型构建[ |
图1 质粒DNA模板在CFPS系统中的研究进展 a:利用质粒DNA体外扩增技术制备大量高纯度DNA,包括RCA与RCR;b:利用高效的分子克隆技术快速制备质粒DNA,包括Gateway克隆系统和GoldenGate克隆系统;c:将质粒DNA与水凝胶结合,如纳米黏土水凝胶和聚N-异丙基丙烯酰胺(pNIPAM)水凝胶,提高DNA局部浓度的同时还可以防止被核酸酶消化;d:质粒DNA来源会影响无细胞蛋白表达水平,应选择与所应用的CFPS系统具有相同细胞背景的表达质粒
Fig. 1 Research progress of plasmid DNA template in CFPS system a:Preparation of large quantities of high-purity DNA by in vitro DNA amplification techniques such as RCA and RCR. b:Rapid preparation of plasmid DNA using efficient cloning techniques,including Gateway cloning system and GoldenGate cloning system. c:Incorporation of plasmids in hydrogels,such as nanoclay hydrogels and poly-N-isopropylacrylamide(pNIPAM)hydrogels,increases the local concentration of DNA while preventing nuclease digestion. d:Plasmid source will affect the level of cell-free protein expression,and an expression plasmid with the same cellular background as the applied CFPS system should be selected
质粒DNA的制备策略 Strategies for preparing plasmid DNA | 特点 Characteristic | 参考文献 Reference | |
---|---|---|---|
体外扩增 In vitro amplification | 滚环扩增RCA | 操作简便、特异性强;成本低 | [ |
复制周期反应RCR | 短时间内获取大量DNA;与从大肠杆菌中获得的质粒相同 | [ | |
高效克隆 Efficient cloning | Gateway | 省时,有助于蛋白高通量表达 | [ |
GoldenGate | DNA组装工具包/试剂盒,加快蛋白功能表征和路径优化 | [ | |
与水凝胶结合 Binding with hydrogel | 黏土水凝胶;基因/黏土/磁性纳米颗粒微凝胶 | 制备简便;DNA局部浓度增加使蛋白产量提高;保护DNA不被核酸酶消化;可以实现蛋白的重复生产 | [ |
pNIPAM水凝胶 | DNA局部浓度增加使蛋白产量提高;转录和翻译过程局限于表面区域,使mRNA水平下降缓慢和蛋白质翻译迅速 | [ | |
质粒DNA来源 Plasmid DNA source | —— | 选择与所应用的CFPS系统具有相同细胞来源的表达质粒会提高蛋白产量 | [ |
表2 质粒DNA模板在CFPS系统中的制备策略
Table 2 Plasmid DNA template preparation strategies in CFPS system
质粒DNA的制备策略 Strategies for preparing plasmid DNA | 特点 Characteristic | 参考文献 Reference | |
---|---|---|---|
体外扩增 In vitro amplification | 滚环扩增RCA | 操作简便、特异性强;成本低 | [ |
复制周期反应RCR | 短时间内获取大量DNA;与从大肠杆菌中获得的质粒相同 | [ | |
高效克隆 Efficient cloning | Gateway | 省时,有助于蛋白高通量表达 | [ |
GoldenGate | DNA组装工具包/试剂盒,加快蛋白功能表征和路径优化 | [ | |
与水凝胶结合 Binding with hydrogel | 黏土水凝胶;基因/黏土/磁性纳米颗粒微凝胶 | 制备简便;DNA局部浓度增加使蛋白产量提高;保护DNA不被核酸酶消化;可以实现蛋白的重复生产 | [ |
pNIPAM水凝胶 | DNA局部浓度增加使蛋白产量提高;转录和翻译过程局限于表面区域,使mRNA水平下降缓慢和蛋白质翻译迅速 | [ | |
质粒DNA来源 Plasmid DNA source | —— | 选择与所应用的CFPS系统具有相同细胞来源的表达质粒会提高蛋白产量 | [ |
图2 CFPS系统中保护线性DNA模板的研究进展 a:对大肠杆菌基因组进行改造,删除内源性核酸酶基因,制备核酸酶缺陷的细胞提取物用于CFPS反应;b:在CFPS反应中添加核酸酶抑制剂如GamS、Chi DNA以及小分子抑制剂,通过抑制剂与RecBCD的直接结合来降低核酸酶活性;c:对线性DNA进行修饰与设计,包括在DNA两端添加保护序列、将DNA交联成水凝胶、将线性DNA连接成环;d:在CFPS反应中添加DNA结合蛋白如Ku、scCro、Tus-Ter,通过与线性DNA的直接结合来保护它不被降解
Fig. 2 Research progress of protecting linear DNA template in CFPS system a:The E. coli genome is modified to delete endogenous nuclease genes,and nuclease-deficient cell extracts are prepared for CFPS reactions. b:Nuclease inhibitors such as GamS,Chi DNA,and small molecule inhibitors are added to the CFPS reaction to reduce the nuclease activity through the direct binding to RecBCD. c:Modification and design of linear DNA template,including adding protection sequences at both ends of DNA,cross-linking DNA into hydrogels,and ligating into DNA circle. d:DNA-binding proteins such as Ku,scCro,Tus-Ter are added to the CFPS reaction to protect linear DNA from degradation
保护线性DNA模板的策略 Strategies for protecting linear DNA templates | 原理 Principles | 蛋白质产量 Protein yield | ||
---|---|---|---|---|
基因组改造 Genomic modifications | ΔrecCBD∷Plac-red-kan-ΔendA[ | 去除提取物中的核酸酶 | 3-6× | 与野生型菌株制备的提取物相比 |
核酸酶抑制剂 Nuclease inhibition | GamS[ | 与RecCBD结合 | 37.6% | 与质粒模板相比 |
小分子抑制剂[ | 作用于解旋酶或ATP酶从而抑制核酸酶活性 | 200% | 与未添加核酸酶抑制剂相比 | |
Chi DNA[ | 与RecCBD结合 | 23% | 与质粒模板相比 | |
修饰和设计线性DNA模板 Modifications and design | 添加poly(G)序列; 添加T7终止序列[ | 增强mRNA稳定性 | 92%(poly(G)) 265%(T7终止子) | 与未修饰的线性DNA模板相比 |
末端添加保护序列[ | 防止核酸酶与线性DNA末端结合 | 75% | 与质粒模板相比 | |
水凝胶[ | 防止被核酸酶降解 | 300% | 与液相体系相比 | |
线性DNA成环[ | 防止被核酸酶降解 | 100% | 与质粒模板相比 | |
DNA结合蛋白 DNA-binding protein | Ku[ | 与线性DNA模板结合 | 4.43×(B. subtilis) 1.58×(C. glutamicum) | 与未添加DNA结合蛋白相比 |
scCro[ | 与线性DNA模板结合 | 6× 18×(V. natriegens) | 与未添加DNA结合蛋白相比 | |
Tus-Ter[ | Tus蛋白与带有Ter序列的线性DNA模板结合 | 156% 164%(V. natriegens) | 与质粒模板相比 |
表3 CFPS系统中稳定线性DNA模板的策略比较
Table 3 Comparison of different linear DNA template protection strategies in CFPS system
保护线性DNA模板的策略 Strategies for protecting linear DNA templates | 原理 Principles | 蛋白质产量 Protein yield | ||
---|---|---|---|---|
基因组改造 Genomic modifications | ΔrecCBD∷Plac-red-kan-ΔendA[ | 去除提取物中的核酸酶 | 3-6× | 与野生型菌株制备的提取物相比 |
核酸酶抑制剂 Nuclease inhibition | GamS[ | 与RecCBD结合 | 37.6% | 与质粒模板相比 |
小分子抑制剂[ | 作用于解旋酶或ATP酶从而抑制核酸酶活性 | 200% | 与未添加核酸酶抑制剂相比 | |
Chi DNA[ | 与RecCBD结合 | 23% | 与质粒模板相比 | |
修饰和设计线性DNA模板 Modifications and design | 添加poly(G)序列; 添加T7终止序列[ | 增强mRNA稳定性 | 92%(poly(G)) 265%(T7终止子) | 与未修饰的线性DNA模板相比 |
末端添加保护序列[ | 防止核酸酶与线性DNA末端结合 | 75% | 与质粒模板相比 | |
水凝胶[ | 防止被核酸酶降解 | 300% | 与液相体系相比 | |
线性DNA成环[ | 防止被核酸酶降解 | 100% | 与质粒模板相比 | |
DNA结合蛋白 DNA-binding protein | Ku[ | 与线性DNA模板结合 | 4.43×(B. subtilis) 1.58×(C. glutamicum) | 与未添加DNA结合蛋白相比 |
scCro[ | 与线性DNA模板结合 | 6× 18×(V. natriegens) | 与未添加DNA结合蛋白相比 | |
Tus-Ter[ | Tus蛋白与带有Ter序列的线性DNA模板结合 | 156% 164%(V. natriegens) | 与质粒模板相比 |
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