第一部分:單核細(xì)胞增殖李斯特菌Imo2088蛋白的晶體結(jié)構(gòu)及性質(zhì)探究單核細(xì)胞增殖李斯特菌（Listeria monocytogenes,LM）是一種食源性的胞內(nèi)寄生革蘭氏陽(yáng)性致病菌,依靠其約200個(gè)調(diào)控基因組成的復(fù)雜網(wǎng)絡(luò)能夠?qū)ν獠凯h(huán)境產(chǎn)生高度適應(yīng)性。這些調(diào)控蛋白被進(jìn)一步分類為不同的家族。其中,Tet R/Acr家族調(diào)控因子在不同基因的調(diào)控中起到至關(guān)重要的作用。Lmo2088基因是TetR/Acr家族的一員,通過(guò)下游基因lmo2087編碼的MATE外排泵調(diào)節(jié)多藥和有毒化合物的外排。本研究的重點(diǎn)是單核增生李斯特菌中推定TetR/AcrR家族轉(zhuǎn)錄調(diào)控因子lmo2088的結(jié)構(gòu)解析。將編碼帶有C端6XHis標(biāo)簽的lmo2088蛋白質(zhì)全長(zhǎng)序列克隆到NdeI和XhoI酶切的pET30a表達(dá)載體上,構(gòu)建載體轉(zhuǎn)化到DH5 α菌株中進(jìn)行復(fù)制,之后轉(zhuǎn)化到BL21和Rosetta菌株中進(jìn)行蛋白表達(dá)。Lmo2088蛋白使用親和層析和凝膠排阻層析法分離純化。純度均一的蛋白使用氣相擴(kuò)散懸滴法進(jìn)行晶體篩選,在進(jìn)行結(jié)晶條件優(yōu)化后獲得單晶晶體,并進(jìn)行衍射數(shù)據(jù)收集。X射線衍射數(shù)據(jù)收集于上海同步輻射光源。晶體衍射分辨率為1.... 

【文章頁(yè)數(shù)】：173 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
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



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