膜蛋白的可溶性表达设计与尝试

摘要膜蛋白是生物膜功能的主要承担者，其结构和功能的研究一直被其低丰度和不溶性所阻碍。我们的目的是通过膜蛋白和增溶蛋白、截断人类载脂蛋白ApoAI（ApoAI*）的融合实现其在水相的可溶性表达。为了达到这一目标，本设计在大肠杆菌耐药因子EmrE上展开探索。通过分子克隆的方法，我们将大肠杆菌耐药因子EmrE和增溶蛋白ΔspMBP、截断人类载脂蛋白ApoAI（ApoAI*）进行融合，构建三元融合表达载体，并将载体转入大肠杆菌表达菌株内进行EmrE的可溶性表达。通过小量诱导表达，细胞破碎和分离等操作，三元融合载体表达并得到水溶性蛋白。本设计提供一种制备可溶性膜蛋白的方法，我们猜想：利用该技术，我们能获得大量的膜蛋白，进而研究膜蛋白的结构和功能。该技术为膜蛋白的研究奠定了基础。42768

关键词大肠杆菌耐药因子EmrE 增溶蛋白ΔspMBP 截断人类载脂蛋白ApoAI（ApoAI*）可溶性表达

毕业论文设计说明书外文摘要

Title design and attempt in soluble expression of membrane proteins

Abstract

Membrane proteins mediate most of the cell functions, whose functional and structural studies are hindered by their low abundance and insolubility. Affinity tag and truncated human apolipoprotein ApoAI (ApoAI*) were fused to the N terminal and C terminal of the membrane protein respectively to get the membrane protein expressed in aqueous phase as a soluble form. In order to achieve this goal, the design carry out an explorations into E. coli multidrug resistance protein E (EmrE).By molecular cloning methods, E. coli multidrug resistance protein E (EmrE) fused with Solubilization protein ΔspMBP at N terminus and truncated human apolipoprotein ApoAI (ApoAI*)at C terminus. Finally a ternary fusion expression vector was constructed, and transformed into E. coli expression strain to attempt the soluble expression of EmrE. After induction, cell fragmentation and separation，a ternary fusion expression vector was expressed and water soluble protein was obtained. The design provided a method for preparing soluble membrane proteins. We suspected that we can get a large amount of membrane proteins using this strategy, thus facilitating further studies of the structure and function of membrane proteins.

Keywords E. coli multidrug resistance protein E (EmrE) Solubilization protein ΔspMBP truncated human apolipoprotein ApoAI (ApoAI*) soluble expression

1 绪论 1

1.1 生物膜和膜蛋白 1

1.2 膜蛋白研究的科学意义和应用前景 2

1.3 膜蛋白的研究现状 2

1.4 膜蛋白的融合表达技术 2

1.5 截断载脂蛋白ApoAI* 3

1.6 增溶蛋白质ΔspMBP 3

1.7 本课题原理及拟研究内容 4

2材料和方法 5

2.1 材料 5

2.1.1 质粒 5

2.1.2 菌种 5

2.1.3 工具酶和试剂盒 5

2.1.4 试剂 5

2.1.5 缓冲液及其他常用溶液 6

2.1.6 培养基 9

2.1.7 主要仪器与设备 9

2.2方法膜蛋白的可溶性表达设计与尝试:http://www.751com.cn/huaxue/lunwen_43316.html