无细胞体系高效合成复杂膜蛋白的关键技术及工业应用探索
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摘要
膜蛋白由于具有极其重要的生物学和医学意义,其研究已经成为蛋白质科学、结构生物学以及医学药学领域的重要部分,同时也是仿生膜等工程领域的研究基础。膜蛋白有不同的分类方法,既包括一次跨膜结构的简单膜蛋白,又包括多次跨膜的复杂膜蛋白。复杂膜蛋白包括受体蛋白,离子通道蛋白,转运蛋白,催化膜上反应的酶等具有各种生物学功能的蛋白质,其结构和功能研究关系着生命科学的各个方面。然而,膜蛋白尤其是复杂膜蛋白的大量制备仍然是现有体内蛋白表达技术难以攻克的难关之一。无细胞蛋白合成体系则为体内难以表达的膜蛋白研究提供了崭新的思路。
本文选取线虫嗅觉受体ODR-10为目标开展了G蛋白偶联受体(GPCR)在无细胞系统高效表达的研究。GPCR是膜蛋白的一个重要组成部分,是许多药物筛选的靶点。本文采取了添加去污剂和脂质体两种策略在大肠杆菌无细胞体系实现ODR-10蛋白的可溶表达,可溶性ODR-10的表达水平达到100μg/mL以上。添加脂质体的策略促进了受体蛋白更加正确的折叠,同时可以使嗅觉受体在原位被合成、折叠和重构,这将方便嗅觉受体蛋白的结构和功能研究,为嗅觉传感器的制备提供支持。
在实现受体蛋白大量可溶表达的基础上,本文进一步研究了通道蛋白的大量制备。选取了大肠杆菌水通道蛋白AqpZ为目标蛋白,利用大肠杆菌无细胞体系高效表达,并采取Ni2+亲和层析纯化得到目标蛋白。纯化后的AqpZ成功嵌入到脂质体上,并证实了功能活性。采用信号肽策略使AqpZ在无细胞体系的表达水平进一步提高。通过在无细胞体系中添加一定浓度的去污剂或脂质体可以原位切除信号肽,简化了纯化步骤,得到了天然大小的具有功能活性的AqpZ。该策略使得在一个反应器中能同时完成转录翻译、信号肽切除、膜蛋白正确折叠的过程。
膜蛋白有许多有前景的工业应用,例如各种仿生膜的制备。在得到大量AqpZ的前提下,选取AqpZ进行了水通道过滤芯片的制备探索。本文制备了纳米氧化铝支撑的双分子膜,并合成了两亲性双嵌段聚合物PMOXA-PDMS-PMOXA,研究了其与AqpZ的相互作用。结果证明聚合物更适于制备稳定的固体支撑膜,用于海水淡化或者废水回收。
许多膜蛋白都具有糖链,大部分有医学应用前景的蛋白质也都是糖蛋白。如何实现糖蛋白的高效异源表达也是膜蛋白大量制备需要解决的问题。本文探索了酵母无细胞蛋白合成体系的制备,发明了一种简便易行的酵母无细胞表达体系。该体系细胞容易培养,成本廉价,制备工艺简单,与其他真核体系相比具有一定优势。通过代谢改造可能会使酵母无细胞系统表达的异源糖蛋白更接近药物蛋白的要求。
总之,本文致力研究膜蛋白在无细胞蛋白合成体系的高效表达策略以及应用研究,并为试图构建新的无细胞蛋白合成体系以适于各种膜蛋白表达。
Membrane proteins have been important research targets in protein science, structural biology, pharmaceuticals, and the preparation of biomimetic membrane, due to their significance in biology and medicine. Membrane proteins include both simple membrane proteins with one transmembrane domain and complex membrane proteins with several transmembrane domains. The structure and function researches of complex membrane proteins, including receptors, ion channel proteins, transporters, and enzymes, are involved in various areas of life sciences. However, high-level production of membrane protein in vivo is still challenging, especially for complex membrane proteins. Cell-free protein synthesis system has attracted many attentions as an alternative tool for the production of membrane proteins.
The olfactory receptor ODR-10from Caenorhabditis elegans, one member of GPCRs, which are usually targets of drug screening, was investigated in E. coli cell-free system. Both addition of detergents and supplementation of liposomes brought about more than100μg/ml soluble ODR-10. The supplied liposomes could integrate protein synthesis, folding and reconstitution in situ, which will facilitate the structure and function studies and pave the application of biosensors.
Besides the large scale production of receptors, the effective expression of channel proteins was investigated. The water channel protein AqpZ was produced in E. coli cell-free system. The protein was effectively expressed and purified by Ni2+affinity chromatography. AqpZ was reconstituted into liposomes successfully and verified to be active. Several natural leader peptides were respectively fused at the N-terminus and verified to enhance the expression level significantly. The supplementation of detergents or liposome could remove the leader peptide in situ, which could simplify the purification process and provide hydrophobic environment for proper folding of active AqpZ. Using this strategy, the transcription-translation, leader sequence cleavage and membrane protein folding were integrated into a simple process in the cell-free system.
As we know, membrane proteins can be applied in various industry areas, for example, the preparation of biomimetic membranes. Based on the large production of AqpZ, the water channel chip preparation was explored in this study. The AAO supported lipid bilayer was prepared and amphiphilic diblock copolymer PMOXA-PDMS-PMOXA was synthesized. The interaction of AqpZ with PMOXA-PDMS-PMOXA was investigated. The result indicated that the copolymer was more suitable for preparation of biomimetic membrane incorporated with AqpZ to be applied in seawater desalination or water recycling.
Many membrane proteins are attached to sugar chains, and most promising targets in therapeutic application are glycoproteins. How to achieve heterologous glycoproteins is a challenging problem. Thus a simple and effective cell-free system based on yeast was established. Comparing to other eukaryotic cell-free systems, the culturing of yeast cells is low-cost, and the yeast cell-free system is easily prepared. The system can be developed to be suitable for production of glycoproteins which can meet the request of pharmaceutical use by metabolic engineering.
This thesis is committed to study the effective production of complex membrane proteins in cell-free system and their applications, as well as the novel cell-free systems investigation for production of various kinds of membrane proteins.
本文选取线虫嗅觉受体ODR-10为目标开展了G蛋白偶联受体(GPCR)在无细胞系统高效表达的研究。GPCR是膜蛋白的一个重要组成部分,是许多药物筛选的靶点。本文采取了添加去污剂和脂质体两种策略在大肠杆菌无细胞体系实现ODR-10蛋白的可溶表达,可溶性ODR-10的表达水平达到100μg/mL以上。添加脂质体的策略促进了受体蛋白更加正确的折叠,同时可以使嗅觉受体在原位被合成、折叠和重构,这将方便嗅觉受体蛋白的结构和功能研究,为嗅觉传感器的制备提供支持。
在实现受体蛋白大量可溶表达的基础上,本文进一步研究了通道蛋白的大量制备。选取了大肠杆菌水通道蛋白AqpZ为目标蛋白,利用大肠杆菌无细胞体系高效表达,并采取Ni2+亲和层析纯化得到目标蛋白。纯化后的AqpZ成功嵌入到脂质体上,并证实了功能活性。采用信号肽策略使AqpZ在无细胞体系的表达水平进一步提高。通过在无细胞体系中添加一定浓度的去污剂或脂质体可以原位切除信号肽,简化了纯化步骤,得到了天然大小的具有功能活性的AqpZ。该策略使得在一个反应器中能同时完成转录翻译、信号肽切除、膜蛋白正确折叠的过程。
膜蛋白有许多有前景的工业应用,例如各种仿生膜的制备。在得到大量AqpZ的前提下,选取AqpZ进行了水通道过滤芯片的制备探索。本文制备了纳米氧化铝支撑的双分子膜,并合成了两亲性双嵌段聚合物PMOXA-PDMS-PMOXA,研究了其与AqpZ的相互作用。结果证明聚合物更适于制备稳定的固体支撑膜,用于海水淡化或者废水回收。
许多膜蛋白都具有糖链,大部分有医学应用前景的蛋白质也都是糖蛋白。如何实现糖蛋白的高效异源表达也是膜蛋白大量制备需要解决的问题。本文探索了酵母无细胞蛋白合成体系的制备,发明了一种简便易行的酵母无细胞表达体系。该体系细胞容易培养,成本廉价,制备工艺简单,与其他真核体系相比具有一定优势。通过代谢改造可能会使酵母无细胞系统表达的异源糖蛋白更接近药物蛋白的要求。
总之,本文致力研究膜蛋白在无细胞蛋白合成体系的高效表达策略以及应用研究,并为试图构建新的无细胞蛋白合成体系以适于各种膜蛋白表达。
Membrane proteins have been important research targets in protein science, structural biology, pharmaceuticals, and the preparation of biomimetic membrane, due to their significance in biology and medicine. Membrane proteins include both simple membrane proteins with one transmembrane domain and complex membrane proteins with several transmembrane domains. The structure and function researches of complex membrane proteins, including receptors, ion channel proteins, transporters, and enzymes, are involved in various areas of life sciences. However, high-level production of membrane protein in vivo is still challenging, especially for complex membrane proteins. Cell-free protein synthesis system has attracted many attentions as an alternative tool for the production of membrane proteins.
The olfactory receptor ODR-10from Caenorhabditis elegans, one member of GPCRs, which are usually targets of drug screening, was investigated in E. coli cell-free system. Both addition of detergents and supplementation of liposomes brought about more than100μg/ml soluble ODR-10. The supplied liposomes could integrate protein synthesis, folding and reconstitution in situ, which will facilitate the structure and function studies and pave the application of biosensors.
Besides the large scale production of receptors, the effective expression of channel proteins was investigated. The water channel protein AqpZ was produced in E. coli cell-free system. The protein was effectively expressed and purified by Ni2+affinity chromatography. AqpZ was reconstituted into liposomes successfully and verified to be active. Several natural leader peptides were respectively fused at the N-terminus and verified to enhance the expression level significantly. The supplementation of detergents or liposome could remove the leader peptide in situ, which could simplify the purification process and provide hydrophobic environment for proper folding of active AqpZ. Using this strategy, the transcription-translation, leader sequence cleavage and membrane protein folding were integrated into a simple process in the cell-free system.
As we know, membrane proteins can be applied in various industry areas, for example, the preparation of biomimetic membranes. Based on the large production of AqpZ, the water channel chip preparation was explored in this study. The AAO supported lipid bilayer was prepared and amphiphilic diblock copolymer PMOXA-PDMS-PMOXA was synthesized. The interaction of AqpZ with PMOXA-PDMS-PMOXA was investigated. The result indicated that the copolymer was more suitable for preparation of biomimetic membrane incorporated with AqpZ to be applied in seawater desalination or water recycling.
Many membrane proteins are attached to sugar chains, and most promising targets in therapeutic application are glycoproteins. How to achieve heterologous glycoproteins is a challenging problem. Thus a simple and effective cell-free system based on yeast was established. Comparing to other eukaryotic cell-free systems, the culturing of yeast cells is low-cost, and the yeast cell-free system is easily prepared. The system can be developed to be suitable for production of glycoproteins which can meet the request of pharmaceutical use by metabolic engineering.
This thesis is committed to study the effective production of complex membrane proteins in cell-free system and their applications, as well as the novel cell-free systems investigation for production of various kinds of membrane proteins.
引文
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