基于TAP技术的TAT-PTD相互作用膜蛋白的筛选与鉴定
发布时间：2019-05-23 03:23
【摘要】：人类Ⅰ型免疫缺陷病毒(human immunodeficiency virus-1,HIV-1)的反式转录激活因子(trans-activator of transcription,TAT)是研究最早、最多的细胞穿透肽(cell penetration peptide,CPP)分子之一。因其具有强大的细胞膜穿透能力,能将与其偶联的“货物”带进细胞而不影响其生物学活性,已经成为药物开发和细胞生物学研究的热点。研究者发现,HIV-TAT能够有效引导肽段或者蛋白质穿透细胞膜,其分子中具有蛋白转导作用的最小结构单元是一个富含碱性氨基酸的多肽片断(第47-57位氨基酸,YGRKKRRQRRR),其特点是转导速度快、效率高,与跨膜功能密切相关,被称为蛋白转导结构域(proteintransduction domain,PTD)。 早期对TAT的跨膜转导机制的研究表明,TAT-PTD通过一个非温度依赖、非能量依赖、非受体依赖的非经典内吞方式的动力学跨膜转运进入细胞。推测这一过程与TAT-PTD中的碱性氨基酸(精氨酸和赖氨酸残基)有关,TAT穿过细胞膜的能力与其富含碱性氨基酸的特点密不可分。这些氨基酸均带有强的正电荷,可能通过直接与带负电荷的细胞膜脂类相互作用,在静电作用的参与下直接穿越细胞膜。后来的研究发现,由于TAT携带了大量阳性电荷,可以与细胞表面带有阴性电荷的硫酸乙酰肝素蛋白聚糖(heparan sulfate proteoglycan,HSPG)发生相互间作用从而启动蛋白转导过程。细胞表面的硫酸乙酰肝素(heparan sulfate,HS)很可能是蛋白内化的关键调节分子结构,在HS表达缺陷的细胞,TAT的转导功能明显受到抑制。在细胞培养上清中加入可溶性肝素、HS模拟物或降解HS的酶均能抑制TAT穿透肽融合蛋白的细胞内化。 在细胞的新陈代谢过程中,不断有各种物质进出细胞,这些物质除了小分子物质外,大分子物质及一些颗粒物质都是通过内化的方式进入细胞。内化广泛参与人体各种生理和病理过程。内吞是大分子物质内化的重要方式,是一个复杂的生物学过程。目前,蛋白及肽类等大分子物质的内化途径主要有两条,一是在受体介导的依赖包涵素的内吞途径,是目前已查明的大多数生物大分子使用的内化途径;二是通过胞膜窖介导的非经典内吞途径,它参与了多种细菌和病毒颗粒的内化过程。近期的研究发现,HSPG是广泛存在于哺乳动物几乎所有粘附细胞表面、胞外基质和基底膜的一类糖蛋白,由一个核心蛋白分子与一个或数个糖胺聚糖通过糖苷键共价结合成的复杂大分子,在细胞粘附、增殖与分化以及血管完整性和通透性方面起重要作用。 串联亲和纯化(tandem affinity purification,TAP)技术是由Rigaut等在1999年建立的,其最初的技术路线是依靠分子克隆技术在细胞中表达含有亲和标签的融合蛋白,通过亲和纯化标签与相应的微珠相互作用,在原核细胞中进行诱饵蛋白的分离纯化。近年来,Stratagene公司开发出一套适用于哺乳动物细胞的TAP系统(InterPlay Mammalian TAP System),用于寻找哺乳类动物细胞内相互作用的蛋白质。改良后的系统使用链霉亲和素结合肽(streptavidin-binding peptide,SBP)纯化标签替代了Prot A和烟草蚀纹病毒(tobacco etchvirus,TEV),避免TEV蛋白酶酶切过程中对靶蛋白的影响,减少了大分子物质对蛋白复合体的污染,使分离的流程变得更加简单,通过钙调蛋白结合多肽(calmodulin binding peptide,CBP)和SBP两个串联的亲和纯化标签对诱饵蛋白复合物连续纯化后,经过两步温和的洗脱(EGTA,生物素),减少与诱饵蛋白相互作用的分子的损失,从而获得更接近自然状态且高纯的特定蛋白复合物。这在研究蛋白质相互作用的方法学上获得了巨大的突破,为研究高等真核生物的蛋白质相互作用网络和蛋白质组学提供了新方法。 TAP技术与质谱技术的联合应用,使得大规模分析蛋白质相互作用成为可能,从而向人们展示出细胞内蛋白质之间的相互作用网络图。这种相互作用的网络图是我们准确理解蛋白质功能,揭开细胞生命奥秘的一个重要技术平台。 基于以上的认识,我们利用TAP系统的原理,首先构建了His、CBP、SBP串联纯化标签与增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)及TAT的融合蛋白表达载体,包括His-CBP-SBP-EGFP、His-CBP-SBP-EGFP-TAT、His-CBP-EGFP、His-CBP-EGFP-TAT、His-SBP-EGFP和His-SBP-EGFP-TAT,在BL21(DE_3)宿主菌诱导表达后,利用Ni~(2+)-NTA亲和树脂进行纯化,并得到相应大小的融合蛋白。通过活细胞影像分析系统研究这些融合蛋白在HepG2细胞的跨膜转导功能;借鉴“串联亲和纯化”的思想,建立TAP下拉(pul-down)技术;利用硫酸乙酰肝素的类似物—肝素进行干预,筛选与TAT-PTD相互作用的小鼠肝脏质膜蛋白,结合质谱强大的鉴定能力来寻找与其相互作用的蛋白,为阐述其穿膜机制提供理论依据。 通过以上研究,我们得到以下结果: (1)成功构建了His-CBP-SBP-EGFP、His-CBP-SBP-EGFP-TAT融合蛋白原核表达载体并证实了His-CBP-SBP-EGFP-TAT能够高效转导进入HepG2细胞;这种转导效应且具有时间和浓度的依赖性。 (2)我们意外发现,对照His-CBP-SBP-EGFP具有膜吸附现象。为明确His-CBP-SBP-EGFP产生细胞膜吸附现象的原因,我们在His-CBP-SBP-EGFP、His-CBP-SBP-EGFP-TAT融合蛋白表达载体基础上成功构建了CBP或SBP序列缺失的His-CBP-EGFP、His-CBP-EGFP-TAT、His-SBP-EGFP和His-SBP-EGFP-TAT融合蛋白原核表达载体。在细胞水平比较了这些融合蛋白的跨膜效应,结果发现去除SBP序列的His-CBP-EGFP融合蛋白的绿色荧光有明显的细胞膜吸附现象,而缺失了CBP的His-SBP-EGFP融合蛋白不能与细胞膜结合,从而证实了引入TAP系统的串联亲和纯化标签CBP会造成融合蛋白在细胞中的分布改变,这可能是由于CBP与细胞膜上的某些结构成份发生相互作用所致。 (3)为了避免CBP标签在纯化过程中增加非特异性的蛋白背景,我们以His-SBP串联亲和纯化标签来作为TAT-PTD相互作用蛋白的纯化系统。经过SDS-PAGE分离蛋白复合物的电泳结果显示,His标签下拉得到的与TAT-PTD相互作用的蛋白复合物经过SBP标签第二次下拉之后,纯化背景明显降低,非特异性的蛋白明显减少,并出现一条非常明显的差异蛋白条带。该条带经质谱鉴定,评分高,具有100%可信度,被鉴定为补体成份1,q亚成份结合蛋白(complement component 1,q subcomponent binding protein,Clqbp)。Clqbp分布于细胞线粒体、线粒体基质和质膜上,对于大分子物质的跨膜转运可能具有重要的作用。 总之,基于TAP技术的原理,本研究成功构建了His-CBP-SBP串联亲和纯化标签的绿色荧光融合蛋白原核表达载体,并对该系统进行了优化,确定CBP缺失的His-SBP串联亲和纯化标签具有背景低,特异性高的优势,为进一步研究TAT-PTD的穿膜机制及其细胞定位提供重要的工具。制备高纯度小鼠肝脏细胞膜蛋白,利用His-SBP串联标签TAP系统进行下拉实验,对蛋白复合物经过His、SBP连续两次亲和纯化,获得了背景低、接近天然的与TAT-PTD相互作用的特异蛋白。差异蛋白经过质谱分析,鉴定为Clqbp,这为深入探讨TAT的跨膜转导机制提供了新的思路。
[Abstract]:The trans-activator of trans-activator of transcription (TAT) of human type I immunodeficiency virus (HIV-1) is one of the earliest and most cell-penetrating peptides (CPP). Because of its powerful cell membrane penetration ability, the "cargo" coupled with it can be taken into the cell without affecting its biological activity, and has become the hot spot of drug development and cell biology research. The researchers found that the HIV-TAT can effectively guide the peptide segment or the protein to penetrate the cell membrane, the minimum structural unit with protein transduction in the molecule is a polypeptide fragment rich in basic amino acid (the amino acid of the 47th-57th position, YGRKRRQRRR), and is characterized in that the transduction speed is high and the efficiency is high, It is closely related to the transmembrane function and is referred to as a protein transduction domain (PTD). The early study of the transmembrane transduction mechanism of TAT shows that TAT-PTD is in the form of a non-temperature-dependent, non-energy-dependent, non-receptor-dependent, non-classical endocytosis. It is assumed that this process is related to the basic amino acids (arginine and lysine residues) in the TAT-PTD, the ability of the TAT to pass through the cell membrane and its rich basic amino acid The amino acids have strong positive charges, which may directly cross the cell by interacting directly with the negatively charged cell membrane lipids and under the participation of the electrostatic effect. The subsequent study found that due to the large number of positive charges carried by the TAT, it was possible to interact with the surface of the cell with a negative charge of glycanoglycan (HSPG) to initiate the protein transduction. Hepan sulfinate (HS) of the cell surface is likely to be the key regulatory molecular structure of the internalization of the protein, and the transduction function of the TAT is obviously inhibited in the cell with the defect of HS expression. The enzyme of soluble heparin, HS mimetic or degradation HS can be added to cell culture to inhibit TAT penetrating peptide fusion protein. In the course of the cell's metabolism, there are various substances in and out of the cell, which, in addition to the small molecular substances, are in the form of internalizing the macromolecular substance and some of the particulate matter. To internalize a wide range of physiological and diseases that are involved in the human body The internal swallowing is an important way of the internalization of macromolecular substances, and it is a complex organism. At present, there are two main pathways for internalization of macromolecular substances such as protein and peptide, one is the endocytosis of the receptor-mediated endostatin, the internalization of most of the biological macromolecules currently identified, and the other is the non-classical endocytosis mediated by the cell membrane. The way, it's involved in a variety of bacteria and viral particles. Recent studies have found that HSPG is a class of glycoproteins that are widely present in almost all of the adherent cell surface, extracellular matrix and basement membrane in a mammal, complex macromolecules that are covalently bound by a core protein molecule with one or more glycosaminoglycans through a sugar-bonded bond, Lifting of cellular adhesion, proliferation and differentiation, and vascular integrity and permeability The tandem affinity purification (TAP) technique was established in 1999 by Rigaut et al., the original technical route of which was to express fusion proteins containing affinity tags in cells by molecular cloning techniques, by affinity purification of the label and corresponding micro-organisms. Bead interaction, bait protein in prokaryotic cells In recent years, Stratagene has developed a set of TAP systems (InterPlay Mammal TAP System) for mammalian cells for the search for each other in mammalian cells The modified system uses the streptavidin-binding peptide (SBP) purification label to replace Prot A and the tobacco-altered virus (TEV), so as to avoid the influence of the TEV protease on the target protein during the digestion process, and the protein complex to the macromolecular substance is reduced. The contamination of the body makes the process of separation more simple, and after continuous purification of the bait protein complex through the calmodulin binding polypeptide (CBP) and the SBP, the bait protein complex is subjected to two-step gentle elution (EGTA, (biotin), the loss of molecules that interact with the bait protein is reduced, Protein complex. This has made a great breakthrough in the research of protein interaction, in order to study the protein interaction network and proteomics of higher eukaryotes. The combination of TAP and mass spectrometry allows for large-scale analysis of protein interactions, thus showing the potential for protein-to-cell interaction The network diagram of this interaction is the one that we can accurately understand the function of the protein and to reveal the mystery of the cell's life. Based on the above, we first constructed a fusion protein expression vector including His-CBP-SBP-EGFP, His-CBP-SBP-EGFP-TAT, His-CBP-EGFP, His-CBP-EGFP-TAT, His-SBP-EGFP and His-SBP. -EGFP-TAT, and purified by using a Ni-(2 +)-NTA affinity resin after the expression of the BL21 (DE _ 3) host bacterium, The fusion protein of the corresponding size was studied by living cell image analysis system. The transmembrane transduction of these fusion proteins in HepG2 cells was studied by living cell image analysis system, and the TAP-down technique was established by using the thought of "tandem affinity purification". The mouse liver membrane protein, combined with the powerful identification ability of the mass spectrum to find the protein interacting with it, in order to set forth its wear The film mechanism provides a theoretical basis. The results are as follows: (1) His-CBP-SBP-EGFP, His-CBP-SBP-EGFP-TAT fusion protein prokaryotic expression vector is successfully constructed and the His-CBP-SBP-EGFP-TAT can be efficiently transduced into HepG2 cells; the transduction effect should have time and concentration dependence. (2) We have surprisingly found that the control of His-CBP- On the basis of His-CBP-SBP-EGFP, His-CBP-SBP-EGFP-TAT fusion protein expression vector, His-CBP-EGFP, His-CBP-EGFP-TAT, His-SBP-EGFP and His-SBP-EGF were successfully constructed on the basis of His-CBP-SBP-EGFP, His-CBP-SBP-EGFP-TAT fusion protein expression vector. P-TAT fusion protein is a prokaryotic expression vector. The transmembrane effect of these fusion proteins is compared with the cell level. The results show that the green fluorescence of the His-CBP-EGFP fusion protein which has removed the SBP sequence has obvious cell membrane adsorption, and the His-SBP-E of the CBP is deleted. The GFP fusion protein cannot be combined with the cell membrane, thus confirming that the serial affinity purification label CBP introduced into the TAP system can cause the distribution change of the fusion protein in the cell, which may be due to the CBP and the cell membrane, (3) In order to avoid the increase of non-specific protein background in the purification process of the CBP label, we use the His-SBP series affinity purification label as the T The purification system of the AT-PTD interaction protein showed that after the second drop-down of the SBP label, the protein complex interacting with the TAT-PTD obtained by the His-tag pull-down, the purified background was significantly reduced and the non-specific protein was significantly reduced. The bands were identified by mass spectrometry, with high scores and 100% confidence, and were identified as complement component 1, q subcomponent binding protein (q subcomponent 1, q subcomponent bind) Clqbp was distributed on the mitochondria, the mitochondrial matrix and the plasma membrane of the cells. In conclusion, based on the principle of TAP technology, this study successfully constructed a prokaryotic expression vector of the green fluorescent fusion protein of His-CBP-SBP tandem affinity purification tag, and optimized the system to determine the His-SBP series of the deletion of CBP. The affinity purification label has the advantages of low background and high specificity, and is a further study of TAT-PT. The membrane protein of the liver cell membrane of the high-purity mouse is prepared, and the pull-down experiment is carried out by using the His-SBP serial tag TAP system, the protein complex is subjected to two-time affinity purification by His and SBP, the background is low, A specific protein that interacts with TAT-PTD. The differential protein is identified as Clqbp by mass spectrometry, which is in-depth
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373
本文编号：2483553
[Abstract]:The trans-activator of trans-activator of transcription (TAT) of human type I immunodeficiency virus (HIV-1) is one of the earliest and most cell-penetrating peptides (CPP). Because of its powerful cell membrane penetration ability, the "cargo" coupled with it can be taken into the cell without affecting its biological activity, and has become the hot spot of drug development and cell biology research. The researchers found that the HIV-TAT can effectively guide the peptide segment or the protein to penetrate the cell membrane, the minimum structural unit with protein transduction in the molecule is a polypeptide fragment rich in basic amino acid (the amino acid of the 47th-57th position, YGRKRRQRRR), and is characterized in that the transduction speed is high and the efficiency is high, It is closely related to the transmembrane function and is referred to as a protein transduction domain (PTD). The early study of the transmembrane transduction mechanism of TAT shows that TAT-PTD is in the form of a non-temperature-dependent, non-energy-dependent, non-receptor-dependent, non-classical endocytosis. It is assumed that this process is related to the basic amino acids (arginine and lysine residues) in the TAT-PTD, the ability of the TAT to pass through the cell membrane and its rich basic amino acid The amino acids have strong positive charges, which may directly cross the cell by interacting directly with the negatively charged cell membrane lipids and under the participation of the electrostatic effect. The subsequent study found that due to the large number of positive charges carried by the TAT, it was possible to interact with the surface of the cell with a negative charge of glycanoglycan (HSPG) to initiate the protein transduction. Hepan sulfinate (HS) of the cell surface is likely to be the key regulatory molecular structure of the internalization of the protein, and the transduction function of the TAT is obviously inhibited in the cell with the defect of HS expression. The enzyme of soluble heparin, HS mimetic or degradation HS can be added to cell culture to inhibit TAT penetrating peptide fusion protein. In the course of the cell's metabolism, there are various substances in and out of the cell, which, in addition to the small molecular substances, are in the form of internalizing the macromolecular substance and some of the particulate matter. To internalize a wide range of physiological and diseases that are involved in the human body The internal swallowing is an important way of the internalization of macromolecular substances, and it is a complex organism. At present, there are two main pathways for internalization of macromolecular substances such as protein and peptide, one is the endocytosis of the receptor-mediated endostatin, the internalization of most of the biological macromolecules currently identified, and the other is the non-classical endocytosis mediated by the cell membrane. The way, it's involved in a variety of bacteria and viral particles. Recent studies have found that HSPG is a class of glycoproteins that are widely present in almost all of the adherent cell surface, extracellular matrix and basement membrane in a mammal, complex macromolecules that are covalently bound by a core protein molecule with one or more glycosaminoglycans through a sugar-bonded bond, Lifting of cellular adhesion, proliferation and differentiation, and vascular integrity and permeability The tandem affinity purification (TAP) technique was established in 1999 by Rigaut et al., the original technical route of which was to express fusion proteins containing affinity tags in cells by molecular cloning techniques, by affinity purification of the label and corresponding micro-organisms. Bead interaction, bait protein in prokaryotic cells In recent years, Stratagene has developed a set of TAP systems (InterPlay Mammal TAP System) for mammalian cells for the search for each other in mammalian cells The modified system uses the streptavidin-binding peptide (SBP) purification label to replace Prot A and the tobacco-altered virus (TEV), so as to avoid the influence of the TEV protease on the target protein during the digestion process, and the protein complex to the macromolecular substance is reduced. The contamination of the body makes the process of separation more simple, and after continuous purification of the bait protein complex through the calmodulin binding polypeptide (CBP) and the SBP, the bait protein complex is subjected to two-step gentle elution (EGTA, (biotin), the loss of molecules that interact with the bait protein is reduced, Protein complex. This has made a great breakthrough in the research of protein interaction, in order to study the protein interaction network and proteomics of higher eukaryotes. The combination of TAP and mass spectrometry allows for large-scale analysis of protein interactions, thus showing the potential for protein-to-cell interaction The network diagram of this interaction is the one that we can accurately understand the function of the protein and to reveal the mystery of the cell's life. Based on the above, we first constructed a fusion protein expression vector including His-CBP-SBP-EGFP, His-CBP-SBP-EGFP-TAT, His-CBP-EGFP, His-CBP-EGFP-TAT, His-SBP-EGFP and His-SBP. -EGFP-TAT, and purified by using a Ni-(2 +)-NTA affinity resin after the expression of the BL21 (DE _ 3) host bacterium, The fusion protein of the corresponding size was studied by living cell image analysis system. The transmembrane transduction of these fusion proteins in HepG2 cells was studied by living cell image analysis system, and the TAP-down technique was established by using the thought of "tandem affinity purification". The mouse liver membrane protein, combined with the powerful identification ability of the mass spectrum to find the protein interacting with it, in order to set forth its wear The film mechanism provides a theoretical basis. The results are as follows: (1) His-CBP-SBP-EGFP, His-CBP-SBP-EGFP-TAT fusion protein prokaryotic expression vector is successfully constructed and the His-CBP-SBP-EGFP-TAT can be efficiently transduced into HepG2 cells; the transduction effect should have time and concentration dependence. (2) We have surprisingly found that the control of His-CBP- On the basis of His-CBP-SBP-EGFP, His-CBP-SBP-EGFP-TAT fusion protein expression vector, His-CBP-EGFP, His-CBP-EGFP-TAT, His-SBP-EGFP and His-SBP-EGF were successfully constructed on the basis of His-CBP-SBP-EGFP, His-CBP-SBP-EGFP-TAT fusion protein expression vector. P-TAT fusion protein is a prokaryotic expression vector. The transmembrane effect of these fusion proteins is compared with the cell level. The results show that the green fluorescence of the His-CBP-EGFP fusion protein which has removed the SBP sequence has obvious cell membrane adsorption, and the His-SBP-E of the CBP is deleted. The GFP fusion protein cannot be combined with the cell membrane, thus confirming that the serial affinity purification label CBP introduced into the TAP system can cause the distribution change of the fusion protein in the cell, which may be due to the CBP and the cell membrane, (3) In order to avoid the increase of non-specific protein background in the purification process of the CBP label, we use the His-SBP series affinity purification label as the T The purification system of the AT-PTD interaction protein showed that after the second drop-down of the SBP label, the protein complex interacting with the TAT-PTD obtained by the His-tag pull-down, the purified background was significantly reduced and the non-specific protein was significantly reduced. The bands were identified by mass spectrometry, with high scores and 100% confidence, and were identified as complement component 1, q subcomponent binding protein (q subcomponent 1, q subcomponent bind) Clqbp was distributed on the mitochondria, the mitochondrial matrix and the plasma membrane of the cells. In conclusion, based on the principle of TAP technology, this study successfully constructed a prokaryotic expression vector of the green fluorescent fusion protein of His-CBP-SBP tandem affinity purification tag, and optimized the system to determine the His-SBP series of the deletion of CBP. The affinity purification label has the advantages of low background and high specificity, and is a further study of TAT-PT. The membrane protein of the liver cell membrane of the high-purity mouse is prepared, and the pull-down experiment is carried out by using the His-SBP serial tag TAP system, the protein complex is subjected to two-time affinity purification by His and SBP, the background is low, A specific protein that interacts with TAT-PTD. The differential protein is identified as Clqbp by mass spectrometry, which is in-depth
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373
【参考文献】
相关期刊论文 前2条
1 揣兰香;硫酸乙酰肝素蛋白聚糖与内科疾病的关系[J];北京医学;2002年03期
2 李海玉;郭爱华;刘志锋;刘瑜;刘靖华;邓鹏;李志杰;刘亚伟;姜勇;;细胞穿透肽核靶向运输蛋白表达载体的构建及其蛋白转导功能的研究[J];南方医科大学学报;2006年10期
，本文编号：2483553
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