融合蛋白Adk-MEK1R4F在大肠杆菌中的表达及应用于ERK蛋白磷酸化的研究

发布时间：2018-06-18 01:12

本文选题：Adk + MEK1　；参考：《西南大学》2017年硕士论文

【摘要】：丝裂原活化蛋白激酶(MAPKs)信号转导通路主要是将细胞外的信号(如生长因子,细胞激素)传递到细胞内及核中从而调控多种细胞反应如生长,分化,炎症和凋亡。目前在哺乳类细胞中已发现存在着三条并行的MAPKs信号通路,包括ERK(extracellular signal-regulated kinase)信号通路,JNK/SAPK信号通路和p38 MAPK信号通路。每一条信号通路均包括三个级联反应蛋白:MAPK,MAPK激酶,MAPK激酶激酶。目前研究得较为全面的是Ras/Raf/MEK/ERK信号通路。丝裂原活化蛋白激酶激酶1(MKK1或者MEK1)是该信号转导通路中的一个重要组成部分,由于其在调节细胞的增殖和分化中的重要作用,MEK1成为了癌症治疗的一个有吸引力的目标。其功能主要是介导下游底物ERK1和ERK2保守性苏氨酸和络氨酸位点的磷酸化。ERK蛋白的磷酸化激活是丝裂原活化的直接反应之一,且ERK自身的磷酸化对ERK蛋白在细胞核中的定位和癌细胞迁移中有重要作用。大多数蛋白激酶活性的激活源于其自身的磷酸化。体外实验中,上游激酶蛋白Mos、MEKK以及大多数的Raf家族蛋白均可以磷酸化MEK1激酶蛋白。然而只有Raf-1是其生理学上的上游激酶蛋白。Raf-1蛋白是由两个功能性不同的结构域组成,两个结构域为氨基端的调控结构域和羧基端的激酶结构域。当将其氨基端结构域敲除后从而变成了组成性激活的催化性结构域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,我们主要是运用MEK1的组成性活性形式MEK1R4F作为研究对象。目前对于人源的MEK1蛋白的研究主要是其表达,纯化以及其激酶特性的研究。然而在以前的研究报道中,MEK1的蛋白表达纯化在昆虫细胞系以及大肠杆菌中均有研究,但是其表达水平非常低也造成后续纯化非常困难,只可以纯化到少量的蛋白且纯度不高。本文主要研究了腺苷酸激酶蛋白作为一个标签蛋白,将其相对应的基因(Adk)通过重叠PCR的方法融合到MEK1R4F基因的N端,此标签蛋白融合后不仅可以增强MEK1R4F在大肠杆菌中的表达水平,还可以促进该蛋白后续的纯化。研究报道,腺苷酸激酶(Adk)是一个重要的磷酸转移酶,其在活细胞从细菌到哺乳动物中均有表达,它在细胞的能量平衡和腺嘌呤核苷酸的代谢中起着重要的作用。源于大肠杆菌的腺苷酸激酶,其不仅在大肠杆菌中有高水平的表达,而且还可以将二磷酸腺苷转换成三磷酸腺苷和腺苷一磷酸。如此融合后的Adk-MEK1R4F蛋白不仅在大肠杆菌中的表达水平大大增加,且通过Ni-NTA的亲和层析以及QFF柱子的阴离子交换层析两步纯化可以得到95%纯度的蛋白。纯化得到的Adk-MEK1R4F融合蛋白不仅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为能量介导ERK蛋白的磷酸化。并且在MEK1R4F利用二磷酸腺苷作为能量介导的ERK磷酸化反应中加入纯化得到的腺苷酸激酶蛋白可以磷酸化ERK。同时我们还发现不管是体外还是体内的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纯化得到更多的磷酸化ERK蛋白。该方法也可以应用于其他重组蛋白的表达,以及其下游纯化。
[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.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q51;Q78

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