大肠杆菌中苹果酸脱氢酶和丝氨酸乙酰转移酶消去化修饰的鉴定

发布时间：2018-04-02 19:34

本文选题：苹果酸脱氢酶　切入点：丝氨酸乙酰转移酶　出处：《西南大学》2017年硕士论文

【摘要】：消去化修饰是近年来发现的一种新的罕见的蛋白质翻译后修饰,是指通过巯基进攻不饱和氨基酸从而发生米歇尔共价加成反应。目前发现的消去化修饰蛋白只有三种,晶状体蛋白、MAPKs和Glutathione peroxidase,而多肽有200多种。对于消去化修饰的鉴定,目前科学界并没有较好的研究方法,为填补这一空白,发现更多的具有消去化修饰的蛋白质及多肽,我们在本实验室已经成熟的标记探针基础上对大肠杆菌BL21(DE3)裂解液进行标记,并鉴定到了221个可能具有消去化修饰的蛋白。选取其中两个大肠杆菌中鉴定到的可能具有消去化修饰的蛋白,苹果酸脱氢酶和丝氨酸乙酰转移酶,进行后期条件更加严格的鉴定。首先通过构建重组菌在大肠杆菌BL21(DE3)中进行诱导表达,选用Ni-TED进行亲和层析纯化得到纯度较高的苹果酸脱氢酶以及丝氨酸乙酰转移酶,利用成熟探针bio-SH试剂对目的蛋白进行初步标记,发现二者具有较好的Michael加成效果。同时为验证所使用的化学探针bio-SH试剂的特异性,排除反应中可能存在非特异性,选择在加成反应过程中加入已验证的具有加成效果的DTT试剂进行竞争,发现苹果酸脱氢酶和丝氨酸乙酰转移酶Michael加成反应有一定程度的减弱,近而说明了化学探针bio-SH试剂具有特异性。其次,将bio-SH试剂进行过化学标记的蛋白与Streptavidin-NHS beads进行室温孵育,对两个目的蛋白进行特异性富集纯化,为后边进行质谱检测奠定了基础。最后,利用CRISPR/Cas9基因编辑技术对大肠杆菌中苹果酸脱氢酶进行敲除,随后通过对其基因缺陷株进行回补以及表达纯化。对目的蛋白进行特异性化学标记后,比较过表达蛋白与正常表达蛋白之间加成效率的差异。其中在缺陷株中蛋白过表达后其加成效率要高于大肠杆菌BL21(DE3)中正常表达的蛋白。本课题建立在本实验室成熟的探针标记基础上,对大肠杆菌中苹果酸脱氢酶和丝氨酸乙酰转移酶进行化学标记,在排除非特异性反应的条件下对二者进行挂柱效率的检测,并通过MDH基因敲除系统的建立,比较蛋白过表达后的加成效率为后边质谱检测以及研究蛋白之间相互作用奠定了基础。同时,该鉴定方法也为后边近一步研究消去化修饰蛋白与多肽的鉴定提供了理论基础。
[Abstract]:Elimination modification is a new rare post-translational modification of proteins discovered in recent years. It refers to the occurrence of Michelle covalent addition reaction through sulfhydryl attack of unsaturated amino acids.At present, there are only three kinds of decontaminated modified proteins, the crystallin MAPKs and Glutathione peroxidase, and the polypeptides are more than 200.In order to fill this gap, more proteins and polypeptides with elimination modification have been found to be more suitable for the identification of decolorization modification.We labeled the cleavage solution of Escherichia coli BL21DDE3 on the basis of a mature labeling probe in our laboratory and identified 221 proteins that may have been modified by elimination.Two proteins identified in E. coli, malate dehydrogenase and serine acetyltransferase, were selected for further identification.The purified malate dehydrogenase and serine acetyltransferase were obtained by Ni-TED affinity chromatography.The mature probe bio-SH reagent was used to label the target protein, and it was found that both of them had good Michael addition effect.In order to verify the specificity of the chemical probe bio-SH reagent used and eliminate the possibility of nonspecificity in the reaction, the addition of DTT reagent with additive effect was selected to compete.It was found that the addition reaction of malate dehydrogenase and serine acetyltransferase Michael was weakened to some extent, which indicated that the chemical probe bio-SH reagent had specificity.Secondly, the proteins labeled with bio-SH reagent were incubated with Streptavidin-NHS beads at room temperature, and the two target proteins were specifically enriched and purified, which laid a foundation for the later detection by mass spectrometry.Finally, CRISPR/Cas9 gene editing technique was used to knockout malate dehydrogenase from Escherichia coli.After specific chemical labeling of the target protein, the addition efficiency difference between the overexpression protein and the normal expression protein was compared.The addition efficiency of protein in defective strain was higher than that in Escherichia coli BL21DE3.Based on the mature probe labeling in our laboratory, the chemical labeling of malate dehydrogenase and serine acetyltransferase in Escherichia coli was carried out.Through the establishment of MDH gene knockout system, the addition efficiency after protein overexpression was compared, which laid a foundation for the detection of protein by back-to-back mass spectrometry and the study of the interaction between proteins.At the same time, the identification method also provides a theoretical basis for the further study on the identification of decolorized modified proteins and peptides.
【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】：Q936

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