拟南芥蛋白sulfiredoxin的重组表达和结晶
发布时间：2022-11-12 11:24
Sulfiredoxin（Srx）作为氧化还原酶家族的成员,在有能量（ATP）存在以及有有机硫化合物并以S-RH形式存在的情况下,特别是当有机硫化合物为硫醇化合物（Cys-SH）时,能够催化过氧化酶的半胱亚磺酸残基向半胱氨酸残基的转化。Srx通过重新活化过氧化物酶而参与抗氧化物的代谢。当peroxiredoxin（Prx）被过氧化抑制时,Srx通过重新还原次磺酸重新激活Prx参与抗氧化代谢。由于有ATP以及Srx存在,半胱氨酸（二硫化物）的这些较低的氧化态和亚磺酸（Cys-SOOH）等较高的氧化态都一样易于逆转。然而,人们对Srx在植物氧化应激状态中的作用了解甚少。本课题的目的是获得重组表达的拟南芥（A.thaliana）的硫氧还蛋白Srx,进行蛋白晶体生长条件筛选和优化,为其结构解析和活性功能分析打下基础。本课题将Srx基因克隆到表达载体pET-22b（+）和pET-28a（+）中,并在大肠杆菌BL21（DE3）表达系统中进行表达。采用镍亲和层析和凝胶过滤对目的蛋白进行纯化,用SDS-PAGE鉴定,获得了纯度较高和状态均一的蛋白。通过晶体生长条件的筛选,在0.8 M NaH...
【文章页数】：35 页
【学位级别】：硕士
【文章目录】：
摘要
abstract
Chapter 1 Introduction
1.1 Oxidative stress in plants:Case of Arabidopsis thaliana
1.1.1 Arabidopsis thaliana:Overview
1.1.2 Oxidative stress in plants:Arabidopsis thaliana
1.1.3 Reactive oxygen species(ROS)and their role in oxidative stress in A.thaliana
1.2 Sulfiredoxin
1.2.1 Structure of Sulfiredoxin
1.2.2 Functions of Sulfiredoxin in A.thaliana
1.2.3 Mutational variation of sulfiredoxin and their functions
Chapter 2 Materials and Methods
2.1 Experimental materials
2.1.1 Experimental strains and vectors
2.1.2 Main reagents in the experiment
2.1.3 Buffer and reagent preparation
2.1.4 Laboratory instruments and equipment
2.2 Clone construction
2.2.1 Amplification of target fragment by PCR
2.2.2 Double digestion of PCR products and vectors
2.2.3 Ligation reaction
2.2.4 Transformation of E.coli competent cells
2.2.5 Recombination verification test
2.3 Protein expression and purification
2.3.1 Protein expression test
2.3.2 Small-scale protein expression
2.3.3 Large-scale protein
2.3.4 Purification of protein by affinity chromatography
2.3.5 Purification of protein by gel filtration chromatography
2.4 Crystal Screen
Chapter 3 Results
3.1 Clone construction
3.2 Recombined protein expression and purification
3.3 Crystal growth conditions of Srx
Chapter 4 Conclusion,discussion and prospect
4.1 Experimental conclusion
4.2 Discussion of recombinant protein Srx expressed on different vectors
4.3 Discussion on the interaction between Srx and Prx
4.4 Prospect of sulfiredoxin
References
Appendix
Acknowledgements
【参考文献】：
期刊论文
[1]Molecular and functional characterization of sulfiredoxin homologs from higher plants[J]. Xian Peng Liu~(1,2) Xue Ying Liu~(1,2) Juan Zhang~(1,2) Zong Liang Xia~(1,2) Xin Liu~1 Huan Ju Qin~1 Dao Wen Wang~11 The State Key Laboratory of Plant Cell and Chromosome Engineering,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Datun Road,Chaoyang District,Beijing 100101,China;2 Graduate University of Chinese Academy of Sciences,Yuquan Road,Shijingshan District,Beijing 100039,China. Cell Research. 2006(03)
本文编号：3706278
【文章页数】：35 页
【学位级别】：硕士
【文章目录】：
摘要
abstract
Chapter 1 Introduction
1.1 Oxidative stress in plants:Case of Arabidopsis thaliana
1.1.1 Arabidopsis thaliana:Overview
1.1.2 Oxidative stress in plants:Arabidopsis thaliana
1.1.3 Reactive oxygen species(ROS)and their role in oxidative stress in A.thaliana
1.2 Sulfiredoxin
1.2.1 Structure of Sulfiredoxin
1.2.2 Functions of Sulfiredoxin in A.thaliana
1.2.3 Mutational variation of sulfiredoxin and their functions
Chapter 2 Materials and Methods
2.1 Experimental materials
2.1.1 Experimental strains and vectors
2.1.2 Main reagents in the experiment
2.1.3 Buffer and reagent preparation
2.1.4 Laboratory instruments and equipment
2.2 Clone construction
2.2.1 Amplification of target fragment by PCR
2.2.2 Double digestion of PCR products and vectors
2.2.3 Ligation reaction
2.2.4 Transformation of E.coli competent cells
2.2.5 Recombination verification test
2.3 Protein expression and purification
2.3.1 Protein expression test
2.3.2 Small-scale protein expression
2.3.3 Large-scale protein
2.3.4 Purification of protein by affinity chromatography
2.3.5 Purification of protein by gel filtration chromatography
2.4 Crystal Screen
Chapter 3 Results
3.1 Clone construction
3.2 Recombined protein expression and purification
3.3 Crystal growth conditions of Srx
Chapter 4 Conclusion,discussion and prospect
4.1 Experimental conclusion
4.2 Discussion of recombinant protein Srx expressed on different vectors
4.3 Discussion on the interaction between Srx and Prx
4.4 Prospect of sulfiredoxin
References
Appendix
Acknowledgements
【参考文献】：
期刊论文
[1]Molecular and functional characterization of sulfiredoxin homologs from higher plants[J]. Xian Peng Liu~(1,2) Xue Ying Liu~(1,2) Juan Zhang~(1,2) Zong Liang Xia~(1,2) Xin Liu~1 Huan Ju Qin~1 Dao Wen Wang~11 The State Key Laboratory of Plant Cell and Chromosome Engineering,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Datun Road,Chaoyang District,Beijing 100101,China;2 Graduate University of Chinese Academy of Sciences,Yuquan Road,Shijingshan District,Beijing 100039,China. Cell Research. 2006(03)
本文编号：3706278
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