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摘要纳米磷脂盘（Nanodisc）系统为膜蛋白的研究提供了一个新平台，利用Nanodisc整合膜蛋白复合物实现气相转移可以进行质谱分析。我们的目的是将虾青素合成途径的膜蛋白酶整合入纳米磷脂盘并利用质谱研究酶活性与其相互作用及构象稳定性之间的关系。为实现这一目标本设计探索了前体茄红素（Lycopene）的表达以及优化: 利用化学转化法将茄红素生长质粒导入大肠杆菌DH5α，使用氯霉素筛选阳性转化菌，提取转化菌株的产物测定其吸收曲线并与番茄红素做对照证明茄红素的产生。以成功导入目标质粒Lycopene的DH5α菌作为表达系统，在不同培养基条件下探究转化菌生长和茄红素生产情况，绘制出转化菌的生长曲线，找出最适合转化菌生长和茄红素生产环境。通过对茄红素生产菌株的优化实验，我们建立了对于底物的定性以及定量分析方法,为后继体外组装形成虾青素合成单元的活性测定打下基础。19524
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关键词 膜蛋白 纳米磷脂盘 质谱分析 茄红素表达系统 虾青素生物合成
毕业论文设计说明书（论文）外文摘要
Title Nanodisc assembly and its application in the structural and functional studies of membrane protein complexs
Abstract
Nanodisc assembly comprising phospholipids (PL) and membrane scaffold proteins (MSP) represents a new platform for the study of the membrane proteins. Utilising the protection of nanodiscs membrane protein complexes can be transferred from solution into the gas phase offering an exciting opportunity for their structural and functional investigation. Based on this concept our overall aim is to incorporate the two enzymes involved in the biogenesis of astaxanthin, CrtW and CrtZ, into nanodiscs for subsequent mass spectrometric analysis, and to correlate the biosynthetic activity with the stoichiometry, interaction and conformational stability of the multienzyme assembly. To the end in this thesis I have carried out a systematic study on the in vivo production of lycopene, the precursor of astaxanthin, and explored various conditions for the optimization of the system: first a lycopene producing plasmid,pLyc,was introduced into E.coli DH5α by chemical transformation. Chloramphenicol was used to screenthe positive transformants. Product of the transformed strain was extracted and its absorption curve detected by a UV-visible spectrophotometer. Using the lycopene standard as a reference we were able to identify the product of the transformed strain. We then explored growth of the transformant and lycopene production under different culture conditions. The growth curves were plotted which enabled us to deduce the most suitable environment for the growth and expression. By establishing the qualitative and quantitative methods for lycopene, these experiments form the basis for the activity assay for the assembly of astaxanthin-producing unit, incorporated in vitro into nanodiscs.
Keywords Membrane proteins; Nanodiscs; Mass spectrometry;Lycopene production; Astaxanthin biogenesis
目 录
1 绪论 1
1.1膜蛋白概述 1
1.1.1膜蛋白的功能与分类 1
1.1.2膜蛋白结构与功能的研究现状 2
1.1.3 虾青素合成途径概述 2
1.2纳米磷脂盘概述 3
1.2.1什么是纳米磷脂盘？ 3
1.2.2纳米磷脂盘的形成 5
1.3纳米磷脂盘体系应用于膜蛋白研究 6
1.3.2通过纳米磷脂盘研究膜蛋白 7