本文選題：Adk + MEK1��； 參考：《西南大學(xué)》2017年碩士論文

【摘要】：絲裂原活化蛋白激酶(MAPKs)信號(hào)轉(zhuǎn)導(dǎo)通路主要是將細(xì)胞外的信號(hào)(如生長(zhǎng)因子,細(xì)胞激素)傳遞到細(xì)胞內(nèi)及核中從而調(diào)控多種細(xì)胞反應(yīng)如生長(zhǎng),分化,炎癥和凋亡。目前在哺乳類(lèi)細(xì)胞中已發(fā)現(xiàn)存在著三條并行的MAPKs信號(hào)通路,包括ERK(extracellular signal-regulated kinase)信號(hào)通路,JNK/SAPK信號(hào)通路和p38 MAPK信號(hào)通路。每一條信號(hào)通路均包括三個(gè)級(jí)聯(lián)反應(yīng)蛋白:MAPK,MAPK激酶,MAPK激酶激酶。目前研究得較為全面的是Ras/Raf/MEK/ERK信號(hào)通路。絲裂原活化蛋白激酶激酶1(MKK1或者M(jìn)EK1)是該信號(hào)轉(zhuǎn)導(dǎo)通路中的一個(gè)重要組成部分,由于其在調(diào)節(jié)細(xì)胞的增殖和分化中的重要作用,MEK1成為了癌癥治療的一個(gè)有吸引力的目標(biāo)。其功能主要是介導(dǎo)下游底物ERK1和ERK2保守性蘇氨酸和絡(luò)氨酸位點(diǎn)的磷酸化。ERK蛋白的磷酸化激活是絲裂原活化的直接反應(yīng)之一,且ERK自身的磷酸化對(duì)ERK蛋白在細(xì)胞核中的定位和癌細(xì)胞遷移中有重要作用。大多數(shù)蛋白激酶活性的激活源于其自身的磷酸化。體外實(shí)驗(yàn)中,上游激酶蛋白Mos、MEKK以及大多數(shù)的Raf家族蛋白均可以磷酸化MEK1激酶蛋白。然而只有Raf-1是其生理學(xué)上的上游激酶蛋白。Raf-1蛋白是由兩個(gè)功能性不同的結(jié)構(gòu)域組成,兩個(gè)結(jié)構(gòu)域?yàn)榘被说恼{(diào)控結(jié)構(gòu)域和羧基端的激酶結(jié)構(gòu)域。當(dāng)將其氨基端結(jié)構(gòu)域敲除后從而變成了組成性激活的催化性結(jié)構(gòu)域Raf-BXB。然而Raf-BXB也是可以激活MEK1蛋白的磷酸化。在本文的研究中,我們主要是運(yùn)用MEK1的組成性活性形式MEK1R4F作為研究對(duì)象。目前對(duì)于人源的MEK1蛋白的研究主要是其表達(dá),純化以及其激酶特性的研究。然而在以前的研究報(bào)道中,MEK1的蛋白表達(dá)純化在昆蟲(chóng)細(xì)胞系以及大腸桿菌中均有研究,但是其表達(dá)水平非常低也造成后續(xù)純化非常困難,只可以純化到少量的蛋白且純度不高。本文主要研究了腺苷酸激酶蛋白作為一個(gè)標(biāo)簽蛋白,將其相對(duì)應(yīng)的基因(Adk)通過(guò)重疊PCR的方法融合到MEK1R4F基因的N端,此標(biāo)簽蛋白融合后不僅可以增強(qiáng)MEK1R4F在大腸桿菌中的表達(dá)水平,還可以促進(jìn)該蛋白后續(xù)的純化。研究報(bào)道,腺苷酸激酶(Adk)是一個(gè)重要的磷酸轉(zhuǎn)移酶,其在活細(xì)胞從細(xì)菌到哺乳動(dòng)物中均有表達(dá),它在細(xì)胞的能量平衡和腺嘌呤核苷酸的代謝中起著重要的作用。源于大腸桿菌的腺苷酸激酶,其不僅在大腸桿菌中有高水平的表達(dá),而且還可以將二磷酸腺苷轉(zhuǎn)換成三磷酸腺苷和腺苷一磷酸。如此融合后的Adk-MEK1R4F蛋白不僅在大腸桿菌中的表達(dá)水平大大增加,且通過(guò)Ni-NTA的親和層析以及QFF柱子的陰離子交換層析兩步純化可以得到95%純度的蛋白。純化得到的Adk-MEK1R4F融合蛋白不僅保留了其完整的磷酸化ERK蛋白激酶活性,而且可以利用除三磷酸腺苷之外的二磷酸腺苷作為能量介導(dǎo)ERK蛋白的磷酸化。并且在MEK1R4F利用二磷酸腺苷作為能量介導(dǎo)的ERK磷酸化反應(yīng)中加入純化得到的腺苷酸激酶蛋白可以磷酸化ERK。同時(shí)我們還發(fā)現(xiàn)不管是體外還是體內(nèi)的ERK磷酸化,Adk-MEK1R4F融合蛋白比非融合MEK1R4F蛋白激酶有更高的活性。因此可以利用此方法去純化得到更多的磷酸化ERK蛋白。該方法也可以應(yīng)用于其他重組蛋白的表達(dá),以及其下游純化。
[Abstract]:Mitogen activated protein kinase (MAPKs) signal transduction pathway is mainly transmitted from extracellular signal (such as growth factor, cell hormone) into cells and nuclei to regulate a variety of cell responses, such as growth, differentiation, inflammation and apoptosis. In mammalian cells, three parallel MAPKs signaling pathways have been found, including ERK (extracell). Ular signal-regulated kinase) signaling pathway, JNK/SAPK signaling pathway and p38 MAPK signaling pathway. Each signal pathway includes three cascade reactive proteins: MAPK, MAPK kinase, and MAPK kinase kinase. The current research is more comprehensive in the Ras/Raf/MEK/ERK signaling pathway. Mitogen activated protein kinase kinase 1 (MKK1 or MEK1) is the signal. An important part of the transduction pathway, as it plays an important role in regulating cell proliferation and differentiation, MEK1 has become an attractive target for cancer treatment. Its function is mainly to mediate phosphorylation of the phosphorylated.ERK protein of the downstream substrate, ERK1 and ERK2 conserved threonine and L-arginine sites, as mitogen activates. The phosphorylation of ERK itself plays an important role in the localization of ERK protein in the nucleus and the migration of cancer cells. Most of the activation of the protein kinase activity is derived from its own phosphorylation. In vitro, the upstream kinase protein Mos, MEKK and most of the Raf family proteins can phosphorylate the MEK1 kinase protein. Only Raf-1 is the physiological upstream kinase protein.Raf-1 protein is composed of two functional domains, two domains are the regulatory domain and the carboxyl terminal kinase domain of the amino terminal domain. When the amino end domain is knocked out, the active domain Raf-BXB. is formed, but Raf-BXB is also In this study, we mainly use the constituent activity of MEK1 as a research object in this study. At present, the research on human MEK1 protein is mainly its expression, purification and the study of its kinase characteristics. However, in previous research, the protein expression of MEK1 was purified in insects. There are studies in cell lines and Escherichia coli, but the expression level is very low and the subsequent purification is very difficult. Only a small amount of protein can be purified and the purity is not high. In this paper, adenylate kinase protein is used as a label protein to fuse its corresponding gene (Adk) into the MEK1R4F gene by overlapping the PCR method. At the N end, the fusion of this tag protein can not only enhance the expression of MEK1R4F in Escherichia coli, but also promote the subsequent purification of the protein. It is reported that adenylate kinase (Adk) is an important phosphoryltransferase, which is expressed in living cells from bacteria to mammals, and it is in cell energy balance and adenine nucleoside. Acid metabolism plays an important role. The adenylate kinase, derived from Escherichia coli, not only has high levels of expression in Escherichia coli, but also converts adenosine two into adenosine triphosphate and adenosine monophosphate. The fusion Adk-MEK1R4F protein not only increases the level of expression in Escherichia coli, but also through Ni-N The purified protein of 95% purity can be obtained by two steps purification of TA affinity chromatography and QFF column anion exchange chromatography. The purified Adk-MEK1R4F fusion protein not only preserves its complete phosphorylated ERK protein kinase activity, but also uses two phosphoric acid adenosine, in addition to adenosine triphosphate, as the phosphorylation of the energy mediated ERK protein. And the adenylate kinase protein that was purified in the MEK1R4F phosphorylation of adenosine two as an energy mediated ERK phosphorylation can be phosphorylated by phosphorylation of ERK., and we also found that Adk-MEK1R4F fusion protein has higher activity than non fusion MEK1R4F protein irritable enzyme in both in vitro and in vivo. Therefore, the Adk-MEK1R4F fusion protein can be used to make use of this side. More phosphorylated ERK protein was purified by the method. The method can also be applied to other recombinant protein expression and downstream purification.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q51;Q78

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本文編號(hào)：2033316