Research Progress of Cell Extracts and Template DNAs in Cell-free Protein Synthesis System

doi:10.13560/j.cnki.biotech.bull.1985.2022-0346

Abstract

Abstract:

Cell-free protein synthesis（CFPS）system is an efficient platform for in vitro protein synthesis. Compared with in vivo systems，it breaks through the limitations of living cells and enables the protein synthesis to be carried out in a test tube，which is flexible，efficient and controllable. In recent years，new technologies in synthetic biology have promoted the rapid development of CFPS system，especially in cell extracts and template DNA. Cell extracts and template DNA are integral components in the CFPS system，largely determining the efficiency and cost of cell-free reactions. This review describes the research progress of cell extracts and template DNA，mainly introduces the characteristics and prospects of newly developed cell extracts，as well as the research progress of plasmid DNA templates and linear DNA templates.

Key words: cell-free protein synthesis, cell extract, plasmid DNA templates, linear DNA templates

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.

Figures/Tables 5

References 127

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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^[36]；生产疟疾蛋白以开发新型疫苗^[37]；研究翻译过程^[38]
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^[39]；核糖体展示技术^[40]
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^[41-42]
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^[43]
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^[44]；生产蛋白质生物制剂^[45]
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^[46]；优化代谢途径^[47]；
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^[48-49]
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^[50]；大规模生产（100 L）^[17]；引入非天然氨基酸^[51]
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^[52]；代谢途径中的瓶颈诊断^[53]
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^[54]；天然产物的生物合成^[55]
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^[56]
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^[57]

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^[36]；生产疟疾蛋白以开发新型疫苗^[37]；研究翻译过程^[38]
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^[39]；核糖体展示技术^[40]
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^[41-42]
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^[43]
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^[44]；生产蛋白质生物制剂^[45]
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^[46]；优化代谢途径^[47]；
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^[48-49]
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^[50]；大规模生产（100 L）^[17]；引入非天然氨基酸^[51]
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^[52]；代谢途径中的瓶颈诊断^[53]
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^[54]；天然产物的生物合成^[55]
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^[56]
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^[57]

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	[92]
体外扩增 In vitro amplification	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	[93]
高效克隆 Efficient cloning	Gateway	省时，有助于蛋白高通量表达	[50]
高效克隆 Efficient cloning	GoldenGate	DNA组装工具包/试剂盒，加快蛋白功能表征和路径优化	[94-95]
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	[96-97]
与水凝胶结合 Binding with hydrogel	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	[98]
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	[57，89]

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	[92]
体外扩增 In vitro amplification	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	[93]
高效克隆 Efficient cloning	Gateway	省时，有助于蛋白高通量表达	[50]
高效克隆 Efficient cloning	GoldenGate	DNA组装工具包/试剂盒，加快蛋白功能表征和路径优化	[94-95]
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	[96-97]
与水凝胶结合 Binding with hydrogel	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	[98]
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	[57，89]

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-Δend^A[110]	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^[107]	与RecCBD结合	37.6%	与质粒模板相比
	小分子抑制剂^[111]	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^[112]	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^[113]	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^[114]	防止核酸酶与线性DNA末端结合	75%	与质粒模板相比
	水凝胶^[102]	防止被核酸酶降解	300%	与液相体系相比
	线性DNA成环^[115]	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^[116]	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^[117]	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^[118]	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