以多巴胺为配基研究蛋白质相互作用的色谱方法
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摘要
细胞中的蛋白质参与和调控着生物体的各种生理活动,其中,大多数蛋白质是通过与配体分子结合或者是作为一个大的生物复合体的一部分来参与细胞生命活动的。因此,研究并阐明蛋白质间相互作用对于理解细胞代谢过程具有重大意义。在实验室前期研究的基础上,本论文继续探索研究蛋白质间相互作用的方法:基于底物与酶的相互作用,以琼脂糖凝胶为载体、乙二胺和戊二醛为间隔臂,固定化多巴胺小分子,吸附以单胺氧化酶B为主的猪肝细胞蛋白质,对单胺氧化酶B与其相关蛋白质间的相互作用进行初步研究。
实验结果表明:(1)通过优化层析材料的配基密度,发现提高多巴胺密度可以增加单胺氧化酶B的吸附量,但其他蛋白量也随之增多,影响单胺氧化酶B纯度。从获得较高的酶比活和酶蛋白纯度的角度出发,配基密度约为36μmol/mL gel的多巴胺层析材料吸附单胺氧化酶B效果比较理想,其洗脱液的酶比活为3723 U/mg,Bandscan软件分析酶蛋白纯度为76%。(2)在采用AKTA凝胶层析对多巴胺层析材料洗脱蛋白进一步分离纯化的过程中,发现单胺氧化酶B(相对分子量约60 KDa)与一个相对分子量约为100 KDa的蛋白质无法完全分离。在进样样品中加入牛血清白蛋白作为参照后,凝胶电泳结果证实单胺氧化酶B与Mr100 KDa蛋白之间存在相互作用。(3)研究比较单胺氧化酶B、猪肝细胞胞质液以及二者混合溶液的酶活性后,发现混合溶液中单胺氧化酶B的酶活性提高了约11倍,初步说明细胞胞质液中含有能够与单胺氧化酶B发生相互作用、促进酶催化活性的物质。
以上研究结果表明:利用固定化多巴胺小分子吸附以单胺氧化酶B为主的猪肝细胞蛋白质,再研究单胺氧化酶B与其相关蛋白间相互作用的方法可行,可作为研究蛋白质间相互作用的色谱方法用于功能蛋白质组学的研究。
The physiological function of organisms is mainly participated and modulated by proteins in cells. Most proteins combined with ligand or form protein complexes with other proteins, and then execute the process of cell metabolism together. Hence, it is significant to elucidate protein-protein interactions for human life. Accroding to the previous research, the purpose of this study is to unceasingly explore the method for protein-protein interaction. To interact with proteins in porcine liver, such as monoamine Oxidase B, a dopamine molecule was immobilized on agarose beads using ethylenediamine and glutaraldehyde as spacer. The interactions between monoamine Oxidase B and its correlative proteins were investigated.
The result of experiments indicated that: (1) It was found that the adsorption capabilities to monoamine Oxidase B were increased by promoting the density of dopamine on support, but the purity of monoamine Oxidase B went up first and then down. As far as higher enzymatic activeness and enzyme purity were concerned, the preferable density of dopamine for purifying monoamine Oxidase B was about 36μmol/mL gel. The activeness of monoamine Oxidase B was 3723 U/mg and its purity was 76% by Bandscan software analyse. (2) After the primary extraction of monoamine Oxidase B from liver crude sample by appropriate density dopamine support, gel filtration was used to progress farther purification. Based on optimization of operating conditions and the control of bovine serum albumin, a protein which may interact with monoamine Oxidase B was found, and its relative molecular weight was about 100000Da. (3) Compared with the purified enzyme eluate, enzymatic activeness of the eluate-cytoplasm mixture increased about eleven times. It primarily showed that some biomolecules in cytoplasm could interact with monoamine Oxidase B and participate in enzymatic catalyze.
All these experiments indicated that it is feasible that a dopamine biomolecule is immobilized on support to interact with monoamine Oxidase B, and the interaction between monoamine Oxidase B and its correlative proteins in cells can be investigated. Therefore, it can be applied in the study of protein-protein interaction in functional proteomics as a new chromatographic method.
实验结果表明:(1)通过优化层析材料的配基密度,发现提高多巴胺密度可以增加单胺氧化酶B的吸附量,但其他蛋白量也随之增多,影响单胺氧化酶B纯度。从获得较高的酶比活和酶蛋白纯度的角度出发,配基密度约为36μmol/mL gel的多巴胺层析材料吸附单胺氧化酶B效果比较理想,其洗脱液的酶比活为3723 U/mg,Bandscan软件分析酶蛋白纯度为76%。(2)在采用AKTA凝胶层析对多巴胺层析材料洗脱蛋白进一步分离纯化的过程中,发现单胺氧化酶B(相对分子量约60 KDa)与一个相对分子量约为100 KDa的蛋白质无法完全分离。在进样样品中加入牛血清白蛋白作为参照后,凝胶电泳结果证实单胺氧化酶B与Mr100 KDa蛋白之间存在相互作用。(3)研究比较单胺氧化酶B、猪肝细胞胞质液以及二者混合溶液的酶活性后,发现混合溶液中单胺氧化酶B的酶活性提高了约11倍,初步说明细胞胞质液中含有能够与单胺氧化酶B发生相互作用、促进酶催化活性的物质。
以上研究结果表明:利用固定化多巴胺小分子吸附以单胺氧化酶B为主的猪肝细胞蛋白质,再研究单胺氧化酶B与其相关蛋白间相互作用的方法可行,可作为研究蛋白质间相互作用的色谱方法用于功能蛋白质组学的研究。
The physiological function of organisms is mainly participated and modulated by proteins in cells. Most proteins combined with ligand or form protein complexes with other proteins, and then execute the process of cell metabolism together. Hence, it is significant to elucidate protein-protein interactions for human life. Accroding to the previous research, the purpose of this study is to unceasingly explore the method for protein-protein interaction. To interact with proteins in porcine liver, such as monoamine Oxidase B, a dopamine molecule was immobilized on agarose beads using ethylenediamine and glutaraldehyde as spacer. The interactions between monoamine Oxidase B and its correlative proteins were investigated.
The result of experiments indicated that: (1) It was found that the adsorption capabilities to monoamine Oxidase B were increased by promoting the density of dopamine on support, but the purity of monoamine Oxidase B went up first and then down. As far as higher enzymatic activeness and enzyme purity were concerned, the preferable density of dopamine for purifying monoamine Oxidase B was about 36μmol/mL gel. The activeness of monoamine Oxidase B was 3723 U/mg and its purity was 76% by Bandscan software analyse. (2) After the primary extraction of monoamine Oxidase B from liver crude sample by appropriate density dopamine support, gel filtration was used to progress farther purification. Based on optimization of operating conditions and the control of bovine serum albumin, a protein which may interact with monoamine Oxidase B was found, and its relative molecular weight was about 100000Da. (3) Compared with the purified enzyme eluate, enzymatic activeness of the eluate-cytoplasm mixture increased about eleven times. It primarily showed that some biomolecules in cytoplasm could interact with monoamine Oxidase B and participate in enzymatic catalyze.
All these experiments indicated that it is feasible that a dopamine biomolecule is immobilized on support to interact with monoamine Oxidase B, and the interaction between monoamine Oxidase B and its correlative proteins in cells can be investigated. Therefore, it can be applied in the study of protein-protein interaction in functional proteomics as a new chromatographic method.
引文
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[2]Kannenberg K,Hunziker P.Interaction between GABA_A Receptor B Subunits and the Multifunctional Protein gClq-R.Journal of Biological Chemistry,2001,276,28:26597-26604.
[3]胡巍,肖志强,陈主初等.鼻咽癌细胞中p53相互作用蛋白质的分离和鉴定.生物化学与生物物理进展,2004,31,7:628-634
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[6]Sui JH,L WH,Murakami A,et al.Potent neutralization of severe acute respiratory syndrome(SARS) coronavirus by a human mAb to S1 protein that blocks receptor association.Proceedings of the National Academy of Sciences of the United State of America,2004,101,8:2536-2541.
[7]Kehoe JW,Velappan N,Walbol M,et al.Using phage display to select antibodies recognizing post-translational modifications independently of sequence context.Molecular & cellular ptroteomics,2006,5,12:2350-2363
[8]Puig O,Caspary F,Rigaut G,et al.The tandem affinity purification(TAP) method:A general procedure of protein complex purification.Methods,2001,24,3:218-229
[9]Arifuzzaman M,Maeda M,Itoh A,et al.Large-scale identification of protein-protein interaction of fschericMa coli K-12.Genome Research,2006,16,5:686-69
[10]Krogan NJ,Cagney G,Yu HY,et al.Global landscape of protein complexes in the yeast Sacharomyces cerevisiae.Nature,2006,440,7084:637-643
[11]Tasto JJ,Carnahan RH,McDonald WH,et al.Vectors and gene targeting modules for tandem affinity purification in Schizosaccharomyces prombe.Yeast,2001,18,7:657-622
[12]Zeghouf M,Li J,Butland G,et al.Sequential peptide affinity(SPA) system for the identification of mammalian and bacterial protein complexes.Journal of Proteome Research,2004,3,3,:463-468
[13]Cheeseman IM,Arshad D.A combined approach for the localization and tandem affinity purification of protein complexes from Metazoans.Science' s STKE,2005,2005,266:p11
[14]Bruckstummer T,Bennett KL,Preradovic A,et al.An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells.Nature Methods,2006,3,12:1013-1019
[15]Rubio V,Shen YP,Saijo Y,et al.An alternative tandem affinity purification strategy applied to Arabidopsis protein complex isolation.Plant Journal,2005,41,5:767-778
[16]Coates PJ,Hall PA.The yeast two-hybrid system for identifying protein-protein interactions.Journal of Pathology,2003,199,1:4-7
[17]Van Rossum DB,Patterson RL,Sharma S,et al.Phospholipase Cy 1 controls surface expression of TRPC3 through an intermolecular PH domain.Nature,2005,434,7029:99-104
[18]刘卉,潘欣,李慧艳等.应用酵母双杂交筛选与CUEDC2相互作用的蛋白质.生物技术通讯,2007,18,5:764-766
[19]Karimova G,Pidoux J,Ullmann A,et al.A bacterial two-hybrid system based on a reconstituted signal transduction pathway.Proceedings of the National Academy of Sciences of the United State of America,1998,95,10:5752-5756
[20]Ladant D,Karimova G.Genetic systems for analyzing protein-protein interactions in bacteria.Research in Microbiology,2000,151,9:711-720
[21]符庆瑛,高钰琪,刘昕.大肠杆菌双杂交系统筛选与AMPKα 2相互作用的蛋白质.第三军医大学学报,2007,29,9:820-823
[22]罗以勤,王梁华,马筱玲等.细菌双杂交系统筛选与tumstatin45-132相互作用蛋白质.中华微生物学和免疫学杂志,2007,27,2:101-106
[23]Leeds JA,Beckwith J.Lambda repressor N-terminal DNA-binding domain as an assay for protein transmembrane segment interaction in vivo.Journal of Molecular Biology,1998,280,5:799-810
[24]Bos JL.An approach involving changes in the emission of fluorescence reveals new information about the activation of two important molecular switches in real time and space.Nature,2001,411,6841:1006-1007
[25]Yan YL,Marriott G.Analysis of protein interactions using fluorescence technologies.Current Opinion in Chemical Biology,2003,7,5:635-640
[26]袁顺宗,谭江琳,彭旭等.荧光共振能量转移技术检测ITGB4BP与P311间的相互作用.第三军医大学学报,2007,29,16:1555-1558
[27]Biener E,Charlier M,Ramanujan VK.Quantitative FRET imaging of leptin receptor oligomerization kinetics in single cells.Biology of the Cell,2005,97,12:905-919
[28]Zhou M,Veenstra TD.Proteomics analysis of protein complexes.Proteomics,2007,7,16:2688-2697
[29]Meng ZJ,Camalier CE,Lucas DA,et al.Probing early growth response 1 interacting proteins at the active promoter in Osteoblast cells using oligoprecipitation and mass spectrometry.Journal of Proteome Research,2006,5,8:1931-1939
[30]Selbach M,Mann M.Protein interaction screening by quantitative immunoprecipitation combined with knockdown(QUICK).Nature Methods,2006,3,12:981-983
[31]Schubert W,Bonnekoh B,Pommer AJ,et al.Analyzing proteome topology and function by automated multidimensional fluorescence microscopy.Nature Biotechnology,2006,24,10:1270-1278
[32]Boguniewicz M,Leung DYM.Atopic dermatitis.Journal of Allergy and Clinical Immunology,2006,117,2:S475-S480
[33]Hu CD,Chinenov Y,Kerppolal TK.Visualization of interactions among bZip and Rel family proteins in living cells using bimolecular fluorescence complementation.Molecular Cell,2002,9,4:789-798
[34]Morell M,Espargaro A,Aviles FX,et al.Detection of transient protein-protein interactions by bimolecular fluorescence complementation:The Ab1-SH3 case.Proteomics,2007,7,7:1023-1036
[35]Hu CD,Kerppola TK.Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis.Nature Biotechnology,2003,21,5:539-545
[36]Grinberg VA,Hu CD,Kerppola TK.Visualization of Myc/Max/Mad family dimmers and the competition for dimerization in living cells.Molecular and Cellular Biology,2004,24,10:4294-4308
[37]Fuentes M,Mateo C,Pessela BCC,et al.Solid phase proteomics:Dramatic reinforcement of very weak protein-protein interactions.Journal of Chromatography B,2007,849,1-2:243-250
[38]Fuentes M,Mateo C,Pessela BCC,et al.Purification,stabilization,and concentration of very weak protein-protein complexes:Shifting the association equilibrium via complex selective adsorption on lowly activated supports.Proteomics,2005,5,16:4062-4069
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