李斯特菌转录调控因子lmo2088和lmo2131的结构研究
发布时间：2023-01-31 05:21
第一部分:单核细胞增殖李斯特菌Imo2088蛋白的晶体结构及性质探究单核细胞增殖李斯特菌（Listeria monocytogenes,LM）是一种食源性的胞内寄生革兰氏阳性致病菌,依靠其约200个调控基因组成的复杂网络能够对外部环境产生高度适应性。这些调控蛋白被进一步分类为不同的家族。其中,Tet R/Acr家族调控因子在不同基因的调控中起到至关重要的作用。Lmo2088基因是TetR/Acr家族的一员,通过下游基因lmo2087编码的MATE外排泵调节多药和有毒化合物的外排。本研究的重点是单核增生李斯特菌中推定TetR/AcrR家族转录调控因子lmo2088的结构解析。将编码带有C端6XHis标签的lmo2088蛋白质全长序列克隆到NdeI和XhoI酶切的pET30a表达载体上,构建载体转化到DH5 α菌株中进行复制,之后转化到BL21和Rosetta菌株中进行蛋白表达。Lmo2088蛋白使用亲和层析和凝胶排阻层析法分离纯化。纯度均一的蛋白使用气相扩散悬滴法进行晶体筛选,在进行结晶条件优化后获得单晶晶体,并进行衍射数据收集。X射线衍射数据收集于上海同步辐射光源。晶体衍射分辨率为1....
【文章页数】：173 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
List of abbreviations and symbols
Chapter 1 Introduction
1.1 Listeria monocytogenes
1.2 Cell infection and invasion of L. monocytogenes
1.3 Intracellular persistence of L. monocytogenes
1.4 Evasion
1.5 Listeriosis
1.6 Listeriosis and clinical features
1.7 What are transcription factors?
1.8 Families of transcriptional regulators
1.8.1. TetR family of transcriptional regulators (TFRs)
1.8.2. The HTH motif
1.9 How TFRs work?
1.10 TFRs and antibiotic resistance
1.11 Homology among TFRs
1.12 Lmo2088-lmo2087 operon
1.13 Protein-DNA interactions
1.13.1. Electrophoretic mobility shift assay (EMSA)
1.13.2. Chromatin immunoprecipitation assays
1.14 Systematic evolution of ligands by exponential enrichment
1.14.1. Random DNA oligonucleotide library
1.14.2. Selection of functional DNA motif sequences
1.14.3. Recovery and identification
1.15 Protein crystallography
1.15.1. Concentration and purity of protein
1.15.2. Protein solubility
1.15.3. Ionic strength
1.15.4. Buffer pH
1.15.5. Additives,effectors,and ligands
1.15.6. Concentration of precipitant
1.15.7. Temperature
1.15.8. Organism source of macromolecules
1.15.9. Metal ions
1.15.10. Reducing or oxidizing environment
1.15.11. Rate of equilibration
1.15.11.1. Vapor diffusion
1.15.11.2. Hanging drop
1.15.11.3. Sitting drop
1.15.11.4. Microbatch
1.15.11.5. Microdialysis
1.16 X-ray diffraction and Crystallography
1.17 Goals and objectives of the project
1.17.1. Structure determination
1.17.2. Functional characterization
Chapter 2 Materials and Methods
2.1 Bacterial strains
2.2 Isolation of genomic DNA from Listeria monocytogenes
2.3 Polymerase chain reaction amplification
2.4 Gel Electrophoresis
2.5 Ethidium bromide (EtBr) staining
2.6 Nucleic acids extraction from gels
2.7 Measurements of DNA concentration
2.8 DNA cloning
2.8.1 Target gene restriction enzyme digestion
2.8.2 Vectors restriction enzyme digestion
2.8.3 Ligation
2.8.4 Transformation
2.8.5 Plasmid confirmation
2.8.6 Plasmid DNA isolation
2.8.7 DNA sequencing
2.9 Small scale induction for protein expression
2.10 Large-scale protein induction
2.11 Affinity chromatography purification
2.12 Removal of DNA from protein sample
2.13 Size exclusion chromatography
2.14 SDS-PAGE
2.15 Non-reducing SDS-PAGE
2.16 Crystallization and X-ray diffraction
2.17 Structure determination procedure
2.18 Protein data bank identification number
2.19 Systematic evolution of ligands by exponential enrichment (SELEX)
2.20 Fluorescence polarization assay
2.21 Electrophoretic mobility shift assay
2.22 Isothermal titration calorimetry
Chapter 3 Results
3.1 Cloning and protein expression on a large-scale
3.2 Protein purification of lmo2088 native protein
3.3 Protein expression and purification of SeMet-lmo2088 protein
3.4 Crystallization
3.5 X-ray diffraction data processing
3.6 Overall structure of the lmo2088
3.7 Dimer interface of lmo2088
3.8 Ligand pocket site of lmo2088
3.9 Sequence alignment of lmo2088
3.10 Structural comparison with TFRs homologs
3.11 DNA binding domain of lmo2088
3.12 Identification of potential DNA oligonucleotide by SELEX
3.13 Sequence analyses SELEX selected DNA
3.14 Fluorescence polarization assay
3.15 EMSA binding interaction
3.16 Binding energetics of lmo2088-y795H1 DNA interaction
Chapter 4 Discussion
4.1. Lmo2088 protein preparations
4.2. TFRs general overview
4.3. Lmo2088 structure
4.4. Lmo2088 potentially binds DNA
4.5. Florescence based Imo2088-DNA complex measurements
4.6. Validation of protein-DNA complexes by EMSA
4.7. Lmo2088 binds cooperatively with 25 bp DNA
4.8. Structural implication of lmo2088
4.9. Conclusions
4.10. Future prospects
References
Chapter 5 Lmo2131 Transcriptional Regulator from Listeria monocytogenes
5.1 Introduction
5.2 Project goals
5.3 Materials and Methods
5.3.1. Strain detail and sequence detail
5.3.2. Cloning of lmo2131 gene
5.3.3. Determination of nucleic acid concentration
5.3.4. Lmo2131 protein preparation
5.3.5. DNA Removal from protein sample
5.3.6. Crystallization and data collection
5.3.7. Systematic evolution of ligands by exponential enrichment (SELEX)
5.4 Results
5.4.1. Molecular cloning
5.4.2. Lmo2131 protein purification
5.4.3. Crystallization
5.4.4. Sequence analysis
5.4.5. Lmo2131 identifies consensus DNA
5.5 Discussion
5.6 Conclusions
5.7 Future Prospect
References
Chapter 6 Appendix
6.1 Primers used in this study
6.2 Chemicals
6.3 Kits
6.4 Detailed bacterial strains
6.5 Vectors
6.6 Antibiotics
6.7 Media
6.8 Buffers
6.9 Laboratory equipment
6.10 The Protein Complex Suite
Acknowledgements
Research outcomes
本文编号：3733749
【文章页数】：173 页
【学位级别】：博士
【文章目录】：
摘要
Abstract
List of abbreviations and symbols
Chapter 1 Introduction
1.1 Listeria monocytogenes
1.2 Cell infection and invasion of L. monocytogenes
1.3 Intracellular persistence of L. monocytogenes
1.4 Evasion
1.5 Listeriosis
1.6 Listeriosis and clinical features
1.7 What are transcription factors?
1.8 Families of transcriptional regulators
1.8.1. TetR family of transcriptional regulators (TFRs)
1.8.2. The HTH motif
1.9 How TFRs work?
1.10 TFRs and antibiotic resistance
1.11 Homology among TFRs
1.12 Lmo2088-lmo2087 operon
1.13 Protein-DNA interactions
1.13.1. Electrophoretic mobility shift assay (EMSA)
1.13.2. Chromatin immunoprecipitation assays
1.14 Systematic evolution of ligands by exponential enrichment
1.14.1. Random DNA oligonucleotide library
1.14.2. Selection of functional DNA motif sequences
1.14.3. Recovery and identification
1.15 Protein crystallography
1.15.1. Concentration and purity of protein
1.15.2. Protein solubility
1.15.3. Ionic strength
1.15.4. Buffer pH
1.15.5. Additives,effectors,and ligands
1.15.6. Concentration of precipitant
1.15.7. Temperature
1.15.8. Organism source of macromolecules
1.15.9. Metal ions
1.15.10. Reducing or oxidizing environment
1.15.11. Rate of equilibration
1.15.11.1. Vapor diffusion
1.15.11.2. Hanging drop
1.15.11.3. Sitting drop
1.15.11.4. Microbatch
1.15.11.5. Microdialysis
1.16 X-ray diffraction and Crystallography
1.17 Goals and objectives of the project
1.17.1. Structure determination
1.17.2. Functional characterization
Chapter 2 Materials and Methods
2.1 Bacterial strains
2.2 Isolation of genomic DNA from Listeria monocytogenes
2.3 Polymerase chain reaction amplification
2.4 Gel Electrophoresis
2.5 Ethidium bromide (EtBr) staining
2.6 Nucleic acids extraction from gels
2.7 Measurements of DNA concentration
2.8 DNA cloning
2.8.1 Target gene restriction enzyme digestion
2.8.2 Vectors restriction enzyme digestion
2.8.3 Ligation
2.8.4 Transformation
2.8.5 Plasmid confirmation
2.8.6 Plasmid DNA isolation
2.8.7 DNA sequencing
2.9 Small scale induction for protein expression
2.10 Large-scale protein induction
2.11 Affinity chromatography purification
2.12 Removal of DNA from protein sample
2.13 Size exclusion chromatography
2.14 SDS-PAGE
2.15 Non-reducing SDS-PAGE
2.16 Crystallization and X-ray diffraction
2.17 Structure determination procedure
2.18 Protein data bank identification number
2.19 Systematic evolution of ligands by exponential enrichment (SELEX)
2.20 Fluorescence polarization assay
2.21 Electrophoretic mobility shift assay
2.22 Isothermal titration calorimetry
Chapter 3 Results
3.1 Cloning and protein expression on a large-scale
3.2 Protein purification of lmo2088 native protein
3.3 Protein expression and purification of SeMet-lmo2088 protein
3.4 Crystallization
3.5 X-ray diffraction data processing
3.6 Overall structure of the lmo2088
3.7 Dimer interface of lmo2088
3.8 Ligand pocket site of lmo2088
3.9 Sequence alignment of lmo2088
3.10 Structural comparison with TFRs homologs
3.11 DNA binding domain of lmo2088
3.12 Identification of potential DNA oligonucleotide by SELEX
3.13 Sequence analyses SELEX selected DNA
3.14 Fluorescence polarization assay
3.15 EMSA binding interaction
3.16 Binding energetics of lmo2088-y795H1 DNA interaction
Chapter 4 Discussion
4.1. Lmo2088 protein preparations
4.2. TFRs general overview
4.3. Lmo2088 structure
4.4. Lmo2088 potentially binds DNA
4.5. Florescence based Imo2088-DNA complex measurements
4.6. Validation of protein-DNA complexes by EMSA
4.7. Lmo2088 binds cooperatively with 25 bp DNA
4.8. Structural implication of lmo2088
4.9. Conclusions
4.10. Future prospects
References
Chapter 5 Lmo2131 Transcriptional Regulator from Listeria monocytogenes
5.1 Introduction
5.2 Project goals
5.3 Materials and Methods
5.3.1. Strain detail and sequence detail
5.3.2. Cloning of lmo2131 gene
5.3.3. Determination of nucleic acid concentration
5.3.4. Lmo2131 protein preparation
5.3.5. DNA Removal from protein sample
5.3.6. Crystallization and data collection
5.3.7. Systematic evolution of ligands by exponential enrichment (SELEX)
5.4 Results
5.4.1. Molecular cloning
5.4.2. Lmo2131 protein purification
5.4.3. Crystallization
5.4.4. Sequence analysis
5.4.5. Lmo2131 identifies consensus DNA
5.5 Discussion
5.6 Conclusions
5.7 Future Prospect
References
Chapter 6 Appendix
6.1 Primers used in this study
6.2 Chemicals
6.3 Kits
6.4 Detailed bacterial strains
6.5 Vectors
6.6 Antibiotics
6.7 Media
6.8 Buffers
6.9 Laboratory equipment
6.10 The Protein Complex Suite
Acknowledgements
Research outcomes
本文编号：3733749
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