基于TAP技術的TAT-PTD相互作用膜蛋白的篩選與鑒定
發(fā)布時間：2019-05-23 03:23
【摘要】：人類Ⅰ型免疫缺陷病毒(human immunodeficiency virus-1,HIV-1)的反式轉錄激活因子(trans-activator of transcription,TAT)是研究最早、最多的細胞穿透肽(cell penetration peptide,CPP)分子之一。因其具有強大的細胞膜穿透能力,能將與其偶聯(lián)的“貨物”帶進細胞而不影響其生物學活性,已經成為藥物開發(fā)和細胞生物學研究的熱點。研究者發(fā)現,HIV-TAT能夠有效引導肽段或者蛋白質穿透細胞膜,其分子中具有蛋白轉導作用的最小結構單元是一個富含堿性氨基酸的多肽片斷(第47-57位氨基酸,YGRKKRRQRRR),其特點是轉導速度快、效率高,與跨膜功能密切相關,被稱為蛋白轉導結構域(proteintransduction domain,PTD)。 早期對TAT的跨膜轉導機制的研究表明,TAT-PTD通過一個非溫度依賴、非能量依賴、非受體依賴的非經典內吞方式的動力學跨膜轉運進入細胞。推測這一過程與TAT-PTD中的堿性氨基酸(精氨酸和賴氨酸殘基)有關,TAT穿過細胞膜的能力與其富含堿性氨基酸的特點密不可分。這些氨基酸均帶有強的正電荷,可能通過直接與帶負電荷的細胞膜脂類相互作用,在靜電作用的參與下直接穿越細胞膜。后來的研究發(fā)現,由于TAT攜帶了大量陽性電荷,可以與細胞表面帶有陰性電荷的硫酸乙酰肝素蛋白聚糖(heparan sulfate proteoglycan,HSPG)發(fā)生相互間作用從而啟動蛋白轉導過程。細胞表面的硫酸乙酰肝素(heparan sulfate,HS)很可能是蛋白內化的關鍵調節(jié)分子結構,在HS表達缺陷的細胞,TAT的轉導功能明顯受到抑制。在細胞培養(yǎng)上清中加入可溶性肝素、HS模擬物或降解HS的酶均能抑制TAT穿透肽融合蛋白的細胞內化。 在細胞的新陳代謝過程中,不斷有各種物質進出細胞,這些物質除了小分子物質外,大分子物質及一些顆粒物質都是通過內化的方式進入細胞。內化廣泛參與人體各種生理和病理過程。內吞是大分子物質內化的重要方式,是一個復雜的生物學過程。目前,蛋白及肽類等大分子物質的內化途徑主要有兩條,一是在受體介導的依賴包涵素的內吞途徑,是目前已查明的大多數生物大分子使用的內化途徑;二是通過胞膜窖介導的非經典內吞途徑,它參與了多種細菌和病毒顆粒的內化過程。近期的研究發(fā)現,HSPG是廣泛存在于哺乳動物幾乎所有粘附細胞表面、胞外基質和基底膜的一類糖蛋白,由一個核心蛋白分子與一個或數個糖胺聚糖通過糖苷鍵共價結合成的復雜大分子,在細胞粘附、增殖與分化以及血管完整性和通透性方面起重要作用。 串聯(lián)親和純化(tandem affinity purification,TAP)技術是由Rigaut等在1999年建立的,其最初的技術路線是依靠分子克隆技術在細胞中表達含有親和標簽的融合蛋白,通過親和純化標簽與相應的微珠相互作用,在原核細胞中進行誘餌蛋白的分離純化。近年來,Stratagene公司開發(fā)出一套適用于哺乳動物細胞的TAP系統(tǒng)(InterPlay Mammalian TAP System),用于尋找哺乳類動物細胞內相互作用的蛋白質。改良后的系統(tǒng)使用鏈霉親和素結合肽(streptavidin-binding peptide,SBP)純化標簽替代了Prot A和煙草蝕紋病毒(tobacco etchvirus,TEV),避免TEV蛋白酶酶切過程中對靶蛋白的影響,減少了大分子物質對蛋白復合體的污染,使分離的流程變得更加簡單,通過鈣調蛋白結合多肽(calmodulin binding peptide,CBP)和SBP兩個串聯(lián)的親和純化標簽對誘餌蛋白復合物連續(xù)純化后,經過兩步溫和的洗脫(EGTA,生物素),減少與誘餌蛋白相互作用的分子的損失,從而獲得更接近自然狀態(tài)且高純的特定蛋白復合物。這在研究蛋白質相互作用的方法學上獲得了巨大的突破,為研究高等真核生物的蛋白質相互作用網絡和蛋白質組學提供了新方法。 TAP技術與質譜技術的聯(lián)合應用,使得大規(guī)模分析蛋白質相互作用成為可能,從而向人們展示出細胞內蛋白質之間的相互作用網絡圖。這種相互作用的網絡圖是我們準確理解蛋白質功能,揭開細胞生命奧秘的一個重要技術平臺。 基于以上的認識,我們利用TAP系統(tǒng)的原理,首先構建了His、CBP、SBP串聯(lián)純化標簽與增強型綠色熒光蛋白(enhanced green fluorescent protein,EGFP)及TAT的融合蛋白表達載體,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)宿主菌誘導表達后,利用Ni~(2+)-NTA親和樹脂進行純化,并得到相應大小的融合蛋白。通過活細胞影像分析系統(tǒng)研究這些融合蛋白在HepG2細胞的跨膜轉導功能;借鑒“串聯(lián)親和純化”的思想,建立TAP下拉(pul-down)技術;利用硫酸乙酰肝素的類似物—肝素進行干預,篩選與TAT-PTD相互作用的小鼠肝臟質膜蛋白,結合質譜強大的鑒定能力來尋找與其相互作用的蛋白,為闡述其穿膜機制提供理論依據。 通過以上研究,我們得到以下結果: (1)成功構建了His-CBP-SBP-EGFP、His-CBP-SBP-EGFP-TAT融合蛋白原核表達載體并證實了His-CBP-SBP-EGFP-TAT能夠高效轉導進入HepG2細胞;這種轉導效應且具有時間和濃度的依賴性。 (2)我們意外發(fā)現,對照His-CBP-SBP-EGFP具有膜吸附現象。為明確His-CBP-SBP-EGFP產生細胞膜吸附現象的原因,我們在His-CBP-SBP-EGFP、His-CBP-SBP-EGFP-TAT融合蛋白表達載體基礎上成功構建了CBP或SBP序列缺失的His-CBP-EGFP、His-CBP-EGFP-TAT、His-SBP-EGFP和His-SBP-EGFP-TAT融合蛋白原核表達載體。在細胞水平比較了這些融合蛋白的跨膜效應,結果發(fā)現去除SBP序列的His-CBP-EGFP融合蛋白的綠色熒光有明顯的細胞膜吸附現象,而缺失了CBP的His-SBP-EGFP融合蛋白不能與細胞膜結合,從而證實了引入TAP系統(tǒng)的串聯(lián)親和純化標簽CBP會造成融合蛋白在細胞中的分布改變,這可能是由于CBP與細胞膜上的某些結構成份發(fā)生相互作用所致。 (3)為了避免CBP標簽在純化過程中增加非特異性的蛋白背景,我們以His-SBP串聯(lián)親和純化標簽來作為TAT-PTD相互作用蛋白的純化系統(tǒng)。經過SDS-PAGE分離蛋白復合物的電泳結果顯示,His標簽下拉得到的與TAT-PTD相互作用的蛋白復合物經過SBP標簽第二次下拉之后,純化背景明顯降低,非特異性的蛋白明顯減少,并出現一條非常明顯的差異蛋白條帶。該條帶經質譜鑒定,評分高,具有100%可信度,被鑒定為補體成份1,q亞成份結合蛋白(complement component 1,q subcomponent binding protein,Clqbp)。Clqbp分布于細胞線粒體、線粒體基質和質膜上,對于大分子物質的跨膜轉運可能具有重要的作用。 總之,基于TAP技術的原理,本研究成功構建了His-CBP-SBP串聯(lián)親和純化標簽的綠色熒光融合蛋白原核表達載體,并對該系統(tǒng)進行了優(yōu)化,確定CBP缺失的His-SBP串聯(lián)親和純化標簽具有背景低,特異性高的優(yōu)勢,為進一步研究TAT-PTD的穿膜機制及其細胞定位提供重要的工具。制備高純度小鼠肝臟細胞膜蛋白,利用His-SBP串聯(lián)標簽TAP系統(tǒng)進行下拉實驗,對蛋白復合物經過His、SBP連續(xù)兩次親和純化,獲得了背景低、接近天然的與TAT-PTD相互作用的特異蛋白。差異蛋白經過質譜分析,鑒定為Clqbp,這為深入探討TAT的跨膜轉導機制提供了新的思路。
[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
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R373
本文編號：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
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R373
【參考文獻】
相關期刊論文 前2條
1 揣蘭香;硫酸乙酰肝素蛋白聚糖與內科疾病的關系[J];北京醫(yī)學;2002年03期
2 李海玉;郭愛華;劉志鋒;劉瑜;劉靖華;鄧鵬;李志杰;劉亞偉;姜勇;;細胞穿透肽核靶向運輸蛋白表達載體的構建及其蛋白轉導功能的研究[J];南方醫(yī)科大學學報;2006年10期
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