摘要腺苷三磷酸结合盒转运蛋白(ATP—binding cassette transporter，ABC转运蛋白)超家族是一组跨膜蛋白，可以在ABC结合域完成单向转运，从而实现物质跨膜运输。我们将基因片段MsbA导入表达载体，目的是在大肠杆菌系统中表达纯化 MsbA蛋白，希望监测其在不同脂类分子和表面活性剂背景下与底物分子的结合的过程，从而建立 MsbA底物竞争性结合的动力学模型。在此过程中利用PCR扩增原理和分子克隆的基本操作来构建出含MsbA基因片段的表达载体，导入最优菌株利用诱导物诱导不同量的菌株表达蛋白。通过用差速离心法与多肽表面活性剂将蛋白稳定的提取出来，并尝试利用咪唑和含His亲和标签的目标蛋白与His柱的亲和竞争关系来纯化蛋白。最后将纯度较高的蛋白浓缩出来得到高浓度蛋白。这将为质谱分析实时监测MsbA构象随结合态的演变提供了材料，有助于我们了解它的外排机制的分子机理。76966

毕业论文关键词：ABC转运蛋白；MsbA；膜蛋白；表达；纯化

毕业设计外文摘要

Title   Dynamics study on the binding of substrates and MsbA,      a ABC transporter                                                   

Abstract The superfamily of ATP binding cassette transporter (ABC transporter) is a group of transmembrane proteins, which can complete unidirectional transport in the ABC binding domain。 We will gene fragment of MsbA into expression vector, the purpose is to make MsbA protein,which will be purified to monitoring the in different lipid molecules and surface active agent in the context and the substrate molecule binds to a process,expressed in Escherichia coli 。so as to establish the MsbA substrate competitive binding to the kinetic model。 In this process we construct expression vector of MsbA gene fragment by polymerase chain reaction (PCR)which is the basic operation principle of molecular cloning and amplification, import the best strain using inducer strains with different amounts of protein expression。 Protein was extracted by differential centrifugation and peptide surfactant, and the protein was purified by the affinity competition between MsbA protein with His tag and imidazole within His trap。 Finally, the high purity of the protein was concentrated to get high concentration of protein。 This will provide a material for mass spectrometry analysis of the evolution of MsbA in real-time monitoring of the conformation of the MsbA protein, which will help us to understand the molecular mechanism of its efflux mechanism。

Keywords：ABC transporter  MsbA  Membrane protein   expression  purification

目   次

1  引言 1

1。1  膜蛋白 1

1。1。1  生物膜与膜蛋白 1

1。1。2  膜蛋白的研究意义 1

1。1。3  膜蛋白的研究现状 2

1。2  ABC转运蛋白 2

1。3  MsbA转运蛋白 3

1。4  MsbA的研究现状 3

1。5  MsbA转运蛋白的纯化策略 3

1。6  ESI质谱分析与动力学研究 4

2  材料和方法 6

2。1  引言 6

2。2  实验材料及仪器