本文選題：蛋白質(zhì)相互作用　切入點(diǎn)：單胺氧化酶B　出處：《大連理工大學(xué)》2010年碩士論文

【摘要】： 蛋白質(zhì)相互作用是功能蛋白質(zhì)組學(xué)研究的重要內(nèi)容,酶作為生物體內(nèi)的一類重要蛋白分子,幾乎參入了所有的生命活動(dòng)和生命過程,研究酶與其它蛋白或調(diào)節(jié)因子的作用將對(duì)人們理解生命體的生理及病理過程具有重要意義。在實(shí)驗(yàn)室前期研究的基礎(chǔ)上,本論文繼續(xù)研究單胺氧化酶B與豬肝細(xì)胞質(zhì)蛋白間相互作用的色譜方法：基于底物與酶的相互作用,通過計(jì)算機(jī)模擬合理設(shè)計(jì)底物配基和間隔臂,優(yōu)化合成材料的配基密度,結(jié)合串聯(lián)質(zhì)譜分析,分子對(duì)接計(jì)算,探索利用固定化底物吸附純化單胺氧化酶B (MAOB)從而鎖定底物與酶的結(jié)合狀態(tài)的可行性。聯(lián)合硫酸銨反抽提、苯胺疏水色譜、強(qiáng)陰離子交換色譜等方法從豬肝線粒體中分離純化出MAOB,進(jìn)一步研究了酶的固定化方法,并對(duì)固定化MAOB與其相關(guān)蛋白質(zhì)間的相互作用進(jìn)行了初步考察。 實(shí)驗(yàn)結(jié)果表明：(1)通過計(jì)算機(jī)對(duì)接模擬,選擇與MAOB有較高親和性的DADPA-戊二醛為連接臂、多巴胺(DA)為配基進(jìn)行材料合成；較高配基密度(40μmol／mL膠)的DA材料可以吸附MAOB,對(duì)其純化倍數(shù)為2.16,但吸附選擇性不好；低密度(10μmol／mL膠)的DA材料分離得到的蛋白較純,經(jīng)串聯(lián)質(zhì)譜鑒定及酶活測(cè)定,不是MAOB,而是以過氧化氫酶為主的蛋白,同時(shí)有少量的肝羧酸酯酶、血清白蛋白；進(jìn)一步的分子對(duì)接實(shí)驗(yàn)說明固定化DA對(duì)MAOB的親和性低于對(duì)過氧化氫酶、肝羧酸酯酶、血清白蛋白的親和力,且固定化DA與MAOB的優(yōu)勢(shì)結(jié)合部位并非酶的活性口袋部位,而是酶表面膜結(jié)合端螺旋結(jié)構(gòu)附近的一處空穴。(2)探索出一種有效分離純化MAOB的方法,即用Triton X-100裂解液制備粗酶、硫酸銨反抽提、苯胺色譜、聯(lián)合Sepharose Q High Performance離子交換色譜,得到純化倍數(shù)為18.2,酶比活為135 U／mg的MAOB。SDS-PAGE顯示為分子量約60 kDa的單一蛋白質(zhì)帶,LC-ESI-MS/MS證實(shí)該蛋白為MAOB。(3)鹽梯度洗脫考察固定化DA材料對(duì)純化的MAOB吸附研究表明,固定化DA與MAOB不能形成穩(wěn)定的吸附,無法進(jìn)一步實(shí)現(xiàn)相互作用蛋白的捕捉和分離。(4)選擇共價(jià)偶聯(lián)法固定MAOB,通過對(duì)連接臂、醛基密度的優(yōu)化,選擇乙二胺、戊二醛為連接臂,醛基密度為5.7μmol／mL膠的材料固定MAOB,固定蛋白量為0.53 mg／mL膠,得到酶活收率為40.98%；MAOB固定化后最適溫度、最適pH升高,溫度及pH穩(wěn)定性增強(qiáng)。(5)用固定化MAOB材料吸附豬肝細(xì)胞質(zhì)蛋白,SDS-PAGE沒有檢測(cè)到相互作用蛋白。 以上研究結(jié)果表明：通過固定化DA吸附純化MAOB并形成底物-酶復(fù)合物的設(shè)想很難實(shí)現(xiàn),且通過SDS-PAGE方法,沒有檢測(cè)到共價(jià)固定的MAOB材料吸附的豬肝細(xì)胞質(zhì)中的相互作用蛋白。
[Abstract]:Protein interaction is an important part of functional proteomics. As a kind of important protein molecules in organism, enzyme is involved in almost all life activities and processes.It is important to study the action of enzyme and other proteins or regulatory factors in understanding the physiological and pathological processes of organisms.Based on the previous research in laboratory, the chromatographic method of interaction between monoamine oxidase B and porcine liver cytoplasmic protein was studied. Based on the interaction between substrate and enzyme, the substrate ligand and spacer were designed by computer simulation.The ligand density of the synthesized materials was optimized, and the binding state of the substrate and the enzyme was determined by using the immobilized substrate to adsorb and purify monoamine oxidase B (MAOB) by means of tandem mass spectrometry and molecular docking calculation.Combined with ammonium sulfate reverse extraction, aniline hydrophobic chromatography and strong anion exchange chromatography, MAOB was isolated and purified from pig liver mitochondria.The interaction between immobilized MAOB and its related proteins was investigated.The results showed that by computer simulation, DADPA-glutaraldehyde, which had high affinity to MAOB, was selected as the connecting arm and dopamine was used as ligand to synthesize.The DA material with a high ligand density of 40 渭 mol/mL) could adsorb MAOB, and its purification multiple was 2.16, but the adsorption selectivity was not good, and the protein isolated from the DA material with low density of 10 渭 mol/mL was relatively pure, which was identified by tandem mass spectrometry and determined by enzyme activity.Not MAOB, but a catalase protein with a small amount of hepatic carboxylesterase and serum albumin. Further molecular docking experiments showed that the affinity of immobilized DA to MAOB was lower than that to catalase and hepatic carboxylesterase.The affinity of serum albumin, and the dominant binding site of immobilized DA to MAOB is not the active pocket site of enzyme, but a hole near the helical structure of enzyme surface membrane.That is, the crude enzyme was prepared from Triton X-100 pyrolysis solution, ammonium sulfate was extracted back, aniline chromatography was used, and Sepharose Q High Performance ion exchange chromatography was used.A single protein band LC-ESI-MS / MS / MS with a specific enzyme activity of 135 U/mg and an enzyme specific activity of 18.2 U/mg was obtained. The protein was confirmed as MAOB.T3 by salt gradient elution. The adsorption of purified MAOB by immobilized DA material was studied.Immobilized DA and MAOB could not form a stable adsorption and could not further realize the capture and separation of interacting proteins. The method of covalent coupling was chosen to immobilize MAOB.According to the optimization of the connecting arm and aldehyde density, ethylenediamine and glutaraldehyde were selected as the connecting arm.The immobilized MAOB was immobilized with a density of 5.7 渭 mol/mL and the amount of protein was 0.53 mg/mL. The optimum temperature and pH of the immobilized MAOB were 40.98% and 40.98 渭 mg/mL, respectively.The interaction protein was not detected by SDS-PAGE when the immobilized MAOB material was used to adsorb porcine liver cytoplasmic protein.The results showed that the idea of purification of MAOB by immobilized DA and the formation of substrate-enzyme complex were difficult to realize, and no interaction protein was detected in porcine liver cytoplasm adsorbed by covalently immobilized MAOB material by SDS-PAGE method.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R341

【引證文獻(xiàn)】

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1 滕婷婷;人IgE受體蛋白FcεRIα-D2的原核表達(dá)及其對(duì)IgE的選擇吸附能力研究[D];大連理工大學(xué);2012年

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本文編號(hào)：1711996