工程化大肠杆菌用于合成甘露糖—6—磷酸和蛛丝蛋白Masp2

论文摘要

甘露糖-6-磷酸（M6P）是一种重要的磷酸糖,其可以参与多种生理功能,在药物合成方面有重要应用。然而,传统方法生产甘露糖-6-磷酸需要用ATP作为磷酸供体,合成成本高昂。为了解决这一问题,在本课题中,我们以甘露糖为底物,使用无机多磷酸盐为磷酸供体,利用甘露糖激酶（glucomannokinase）催化合成M6P,从而避免ATP的使用。我们从草分枝杆菌（Mycobacterium phlei）中克隆出甘露糖激酶基因,并将其转化到大肠杆菌中进行诱导表达,随后获得的酶被纯化。甘露糖激酶蛋白大小30kDa,纯化结果由蛋白电泳检测。最终,我们获得了 0.69mg/mL的甘露糖激酶,其纯度超过90%。动力学研究表明,该酶对多磷酸盐和葡萄糖的催化性能优于ATP和甘露糖,其米氏常数KM值分别为1.7μM,9.5 μM,4.6 μM和203.7 μM。进一步实验表明,甘露糖-6-磷酸生产的最适反应条件是:pH8.5,25℃,多磷酸盐/甘露糖=3:1。另外,一定浓度的Mg2+有助于提高酶活性。结果说明来自草分枝杆菌的葡糖甘露糖酶有工业化生产甘露糖-6-磷酸的潜力。此外,本课题还进行了另一方面的研究——蛛丝蛋白的MaSp2的合成。丝是一种重要的生物产品,其可以用来制作许多重要的生物材料,如缝合线等。一般地,丝含有两种重要的蛋白质,壶腹蛛丝蛋白1和2（MaSp1;MaSp1）。在本课题中,我们尝试使用大肠杆菌BL21（DE3）作为宿主,人工合成MaSp2。起初,我们构建了名为gene unit的丝基因的单体单元（约100bp）。随后,这些单链寡核苷酸gene unit被头尾连接,以得到不同长度的聚合gene unit（聚合度为2-64）。这些聚合gene unit被插入到质粒中。质粒pET28a（+）和pET22b（+）分别带有MaSp2和Ala/glyc-tRNA基因,其被转化到在大肠杆菌BL21（DE3）中共表达。重组大肠杆菌在含有0.05%丙氨酸和甘氨酸的培养基中培养（避免蛋白翻译在早期停止）。最终,我们得到了大小为55和110 kDa的人工MaSp2蛋白。结果说明了 MaSp2能够被人工合成,工程化大肠杆菌可以被用于表达高分子量和具有重复氨基酸序列的蛋白质。

论文目录

• ABSTRACT
• 摘要
• Chapter 1: Introduction
•   1.1 Introduction of biotechnology
•     1.1.1 Agricultural uses
•     1.1.2 Industrial benefits
•     1.1.3 Environmental benefits
•   1.2 Research objective
•   Section Ⅰ
•     1.3 Mannose-6-phosphate background
•       1.3.1 Clinical significance and literature review
•       1.3.2 Synthesis of Mannose-6-phosphate
•       1.3.3 Research objective
•       1.3.4 Glucomannokinase: A Biocatalyst used in this research
•       1.3.5 Inorganic polyphosphate a phosphorylating agent: overview
•   Section Ⅱ
•     1.4 Introduction of silk protein
•       1.4.1 Types of silk proteins
•       1.4.2 The Structure and composition of dragline silk
•       1.4.3 Background of Recombinant silk proteins Production
•       1.4.4 Heterologous Host System used to produce silk protein
•       1.4.5 Strategies to design and assemble the monomeric gene to produce lager cassette
•       1.4.6 Research objective
• Chapter 2: Materials and Methodology
•   2. Introduction
•   Section Ⅰ
•     2.1 Materials and methodology of production of M6P
•       2.1.1 Strains, plasmids, and chemicals
•       2.1.2 Cloning of poly（P）/ATP-hexokinase gene and construction of the expression vector
•       2.1.3 Expression of recombinant poly（P）/ATP-hexokinase
•       2.1.4 Poly（P）/ATP-hexokinase production in E. coli analyzed by SDS-PAGE
•       2.1.5 Purification of poly（P）/ATP-hexokinase
•       2.1.6 Optimization of induction temperature and concentration of IPTG and their effects onenzyme activity
•       2.1.7 Poly（P）/ATP-hexokinase assay
•       2.1.8 Determination of kinetic parameters
•       2.1.9 Analysis of product using high-performance anion-exchange HPLC
•       2.1.10 Effects of various parameters on the poly（P）/ATP-hexokinase activity
•   Section Ⅱ
•     2.2 Materials, Methodology and Experimentation of MaSp2 production
•       2.2.1 Strains, plasmids, and chemicals
•       2.2.2 Cloning and construction of monomeric unitl-64
•       2.2.3 Purification of cloned plasmids
•       2.2.4 Transformation and Expression of recombinant plasmids
•       2.2.5 Expressed Dragline Silk Protein was analyzed by SDS-PAGE
•       2.2.6 Protein purification using chromatography
•       2.2.7 Optimization of translation process
•       2.2.8 Effects amino acids on cell density and protein expression
•       2.2.9 Analysis of composition of expressed proteins
• Chapter 3: Result and discussion of Mannose-6-phosphate production
•   3.1 Glucomannokinase enzyme Sequences alignment using ClustaX
•   3.2 Heterologous Protein expression,purification and analysis
•   3.3 Optimization of induction temperature and concentration of IPTG
•     3.3.1 SDS-PAGE analysis of protein at various temperature and IPTG
•     3.3.2 Effects of induction temperature on biomass, protein content and enzyme activity
•   3.4 Kinetic parameters
•   3.5 Optimization of conditions
•     3.5.1 Optimum pH
•     3.5.2 Optimum temperature
•     3.5.3 Ratio of poly（P）/mannose
•     3.5.4 Ion preference
•     3.5.5 Optimization of Mg2+
•     3.5.6 Optimization of different polyphosphate
•   3.6 Reaction course
•   3.7 summary
• Chapter 4: Result and discussion of Production of Spider silk protein
•   4.1 Designing and assembling of the monomer gene unit （GU） of MaSp2
•   4.2 Construction of basic GU, GU-2n and 4n of MaSp2 by PCR
•   4.3 Construction of GU-8n to 64n of MaSp2
•   4.4 Construction, Expression and Verification of pet28a+GU-32n,pet28a+GU-16n, pet28a+GU-8n.
•     4.4.1 Effect of alanine/glycine on expression of pET28a+GU-16n engineeringbacteria
•     4.4.2 Effects of temperature on induction of pET28a+GU-16n
•     4.4.3 PAGE analysis of the GU-16n protein expression in optimal expression conditions
•   4.5 Expression of pET28a+GU-32n under optimize medium
•   4.6 Expression of the pET28a-Spin32 at enhanced tRNA pool
•   4.7 Effect of amino acids on cell density and protein content of GU-32n
•   4.8 Amino acid analysis
•   4.9 Summary
• Chapter 5 Conclusion
• References
• Acknowledgement
• Research Publication
• Introduction to Author & Supervisor
• 附件

文章来源

类型: 硕士论文

作者: Shafaq Parveen

导师: 刘珞

关键词: 磷酸甘露糖,甘露糖激酶,聚磷酸盐,丝蛋白,壶腹蛛丝蛋白

来源: 北京化工大学

年度: 2019

分类: 基础科学

专业: 生物学

单位: 北京化工大学

分类号: Q78

DOI: 10.26939/d.cnki.gbhgu.2019.000686

总页数: 111

文件大小: 7156K

下载量: 17