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Identification and functional characterisation of a PREP1-PBX protein complexBerthelsen, Jens January 2000 (has links)
No description available.
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DNA Binding and Photocleavage by [Rh2(DPhF)2(bncn)2]2+Wroblewski, Rebekah Abigail January 2021 (has links)
No description available.
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Functional and structural properties of eukaryotic DNA polymerase epsilonChilkova, Olga January 2006 (has links)
In eukaryotes there are three DNA polymerases which are essential for the replication of chromosomal DNA: DNA polymerase alpha (Pol alpha), DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon). In vitro studies of viral DNA replication showed that Pol alpha and Pol delta are sufficient for DNA replication on both leading and lagging DNA strands, thus leaving the function of Pol epsilon unknown. The low abundance and the reported protease sensitivity of Pol epsilon were holding back biochemical studies of the enzyme. The aim of this study was to characterize the structural and functional properties of eukaryotic Pol epsilon. We first developed a protocol for over-expression and purification of Pol epsilon from the yeast Saccharomyces cerevisiae. Pol epsilon consists of four subunits: Pol2 (catalytic subunit), Dpb2, Dpb3 and Dpb4. This four-subunit complex was purified to homogeneity by conventional chromatography and the subunit stoichiometry of purified Pol epsilon was estimated from colloidal coomassie-stained gels to be 1:1:1:1. The quaternary structure was determined by sedimentation velocity and gel filtration experiments. Molecular mass (371 kDa) was calculated from the experimentally determined Stokes radius (74.5 Å) and sedimentation coefficient (11.9 S) and was in good agreement with a theoretical molecular mass calculated for a heterotetramer (379 kDa). Analytical sedimentation equilibrium ultracentrifugation experiments supported the proposed heterotetrameric structure of Pol epsilon. By cryo-electron microscopy and single-particle image analysis we determined the structure of Saccharomyces cerevisiae Pol epsilon to 20-Å resolution. The four-subunit complex was found to consist of a globular domain, comprising the Pol2 subunit, flexibly connected to an elongated domain, including Dpb2, Dpb3 and Dpb4 subunits. We found that Pol epsilon requires a minimal length of 40 base pairs of primer-template duplex to be processive. This length corresponds to the dimensions of the elongated domain. To characterize the fidelity by which Pol epsilon synthesizes DNA, we purified wild type and exonuclease-deficient Pol epsilon. Wild type Pol epsilon synthesizes DNA with a very high accuracy. Analysis of the exonuclease-deficient Pol epsilon showed that Pol epsilon proofreads more than 90% of the errors made by its polymerase activity. Exonuclease-deficient Pol epsilon was shown to have a specific spectrum of errors not seen in other DNA polymerases: a high proportion of transversions resulting from T-dTTP, T-dCTP and C-dTTP mispairs. This unique error specificity and amino acid sequence alignment suggest that the structure of the polymerase active site of Pol epsilon differs from those of other members of B family DNA polymerases. With recombinant proteins and circular single-stranded DNA templates, we partially reconstituted DNA replication in vitro, in which we challenged Pol epsilon and Pol delta in side-by-side comparisons regarding functional assays for polymerase activity and processivity, as well as physical interactions with nucleic acids and PCNA. We found that Pol epsilon activity and “on-DNA” PCNA interactions are dependent on RPA-coated template DNA. By the surface plasmon resonance technique, we showed that Pol epsilon has a high affinity for DNA and low affinity for immobilized PCNA. By contrast, Pol delta was found to have low affinity for DNA and high affinity for PCNA. We suggest that a possible function of RPA is to regulate down the DNA synthesis through Pol epsilon, and that the mechanism by which Pol epsilon and Pol delta load onto the template is different due to different properties of the interaction with DNA and PCNA.
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Structural and Functional Evolution of Human Heat Shock Transcription FactorsJaeger, Alex M. January 2015 (has links)
<p>Proteotoxic stress is implicated in numerous human diseases including neurodegeneration, cancer, and diabetes. Unfortunately, our mechanistic understanding of the cellular response to proteotoxic stress is limited. A critical feature of the cellular stress response is the activation of Heat Shock Transcription Factors (HSFs) that regulate the expression of numerous genes involved in protein folding, protein degradation, and cellular survival. The studies presented here utilize a diverse array of techniques including yeast genetics, recombinant protein expression and purification, biochemical analysis of protein-DNA interactions, x-ray crystallography, in vitro post-translational modification, and mammalian cell culture to illuminate novel aspects of HSF biology. Critical findings include understanding key principles of HSF-DNA interactions, identification of a novel negative regulator of HSF activity, and identification of structural features of HSF paralogs that enable precise combinatorial regulation. These unique insights lay the foundation for a greater understanding of HSF in specific cellular contexts and disease states.</p> / Dissertation
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Large scale simulations of genome organisation in living cellsJohnson, James January 2018 (has links)
Within every human cell, approximately two meters of DNA must be compacted into a nucleus with a diameter of around ten micrometers. Alongside this daunting storage problem, the 3D organisation of the genome also helps determine which genes are up- or down-regulated, which in turn effects the functionality of the cell itself. While the organisational structure of the genome can be revealed using experimental techniques such as chromosome conformation capture and its high-throughput variant Hi-C, the mechanisms driving this organisation are still unclear. The first two results chapters of this thesis use molecular dynamics simulations to investigate the effect of a potential organisational mechanisms for DNA known as the "bridging-induced attraction". This mechanism involves multivalent DNA-binding proteins bridging genomically distant regions of DNA, which in turn promotes further binding of proteins and compaction of the DNA. In chapter 2 (the first results chapter) we look at a model where proteins can bind non-specifically to DNA, leading to cluster formation for suitable protein-DNA interaction strengths. We also show the effects of protein concentration on the DNA, with a collapse from a swollen to a globular phase observed for suitably high protein concentrations. Chapter 3 develops this model further, using genomic data from the ENCODE project to simulate the "specific binding" of proteins to either active (euchromatin) or inactive (heterochromatin) regions. We were then able to compare contact maps for specific simulated chromosomes with the experimental Hi-C data, with our model reproducing well the topologically associated domains (TADs) seen in Hi-C contact maps. In chapter 4 of the thesis we use numerical methods to study a model for the coupling between DNA topology (in particular, supercoiling in DNA and chromatin) and transcription in a genome. We present details of this model, where supercoiling flux is induced by gene transcription, and can diffuse along the DNA. The probability of transcription is also related to supercoiling, as regions of DNA which are negatively supercoiled have a greater likelihood of being transcribed. By changing the magnitude of supercoiling flux, we see a transition between a regime where transcription is random and a regime where transcription is highly correlated. We also find that divergent gene pairs show increased transcriptional activity, along with transcriptional waves and bursts in the highly correlated regime { all these features are associated with genomes of living organisms.
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DNA Mismatch Repair In Haemophilus Influenzae : Characterization Of MutH, L, S And Their InteractionJoseph, Nimesh 12 1900 (has links) (PDF)
No description available.
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Discovery of New Protein-DNA and Protein-Protein Interactions Associated With Wood Development in Populus trichocarpaPetzold, Herman E. III 09 November 2017 (has links)
The negative effects from rising carbon levels have created the need to find alternative energy sources that are more carbon neutral. One such alternative energy source is to use the biomass derived from forest trees to fulfill the need for a renewable alternative fuel. Through increased understanding and optimization of regulatory mechanisms that control wood development the potential exists to increase biomass yield. Transcription factors (TFs) are DNA-binding regulatory proteins capable of either activation or repression by binding to a specific region of DNA, normally located in the 5-prime upstream promoter region of the gene. In the first section of this work, six DNA promoters from wood formation-related genes were screened by the Yeast One-Hybrid (Y1H) assay in efforts to identify novel interacting TFs involved in wood formation. The promoters tested belong to genes involved in lignin biosynthesis, programmed cell death, and cambial zone associated TFs. The promoters were screened against a mini-library composed of TFs expressed 4-fold or higher in differentiating xylem vs phloem-cambium. The Y1H results identified PtrRAD1 with interactions involving several of the promoters screened. Further testing of PtrRAD1 by Yeast Two-Hybrid (Y2H) assay identified a protein-protein interaction (PPI) with poplar DIVARACATA RADIALIS INTERACTING FACTOR (DRIF1). PtrDRIF1 was then used in the Y2H assay and formed PPIs with MYB/SANT domain proteins, homeodomain family (HD) TFs, and cytoskeletal-related proteins. In the second section of this work, PPIs involving PtrDRIF1s' interaction partners were further characterized. PtrDRIF1 is composed of two separate domains, an N-terminal MYB/SANT domain that interacted with the MYB/SANT domain containing PtrRAD1 and PtrDIVARICATA-like proteins, and a C-terminal region containing a Domain of Unknown Function 3755 (DUF3755). The DUF3755 domain interacted with HD family members belonging to the ancient WOX clade and Class II KNOX domain TFs. In addition, PtrDRIF1 was able to form a complex between PtrRAD1 and PtrWOX13c in a Y2H bridge assay. PtrDRIF1 may function as a regulatory module linking cambial cell proliferation, lignification, and cell expansion during growth. Combined, these findings support a role for PtrDRIF1 in regulating aspects of wood formation that may contribute to altering biomass yield. / Ph. D.
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From network to pathway: integrative network analysis of genomic dataWang, Chen 25 August 2011 (has links)
The advent of various types of high-throughput genomic data has enabled researchers to investigate complex biological systems in a systemic way and started to shed light on the underlying molecular mechanisms in cancers. To analyze huge amounts of genomic data, effective statistical and machine learning tools are clearly needed; more importantly, integrative approaches are especially needed to combine different types of genomic data for a network or pathway view of biological systems. Motivated by such needs, we make efforts in this dissertation to develop integrative framework for pathway analysis. Specifically, we dissect the molecular pathway into two parts: protein-DNA interaction network and protein-protein interaction network. Several novel approaches are proposed to integrate gene expression data with various forms of biological knowledge, such as protein-DNA interaction and protein-protein interaction for reliable molecular network identification.
The first part of this dissertation seeks to infer condition-specific transcriptional regulatory network by integrating gene expression data and protein-DNA binding information. Protein-DNA binding information provides initial relationships between transcription factors (TFs) and their target genes, and this information is essential to derive biologically meaningful integrative algorithms. Based on the availability of this information, we discuss the inference task based on two different situations:
(a) if protein-DNA binding information of multiple TFs is available:
based on the protein-DNA data of multiple TFs, which are derived from sequence analysis between DNA motifs and gene promoter regions, we can construct initial connection matrix and solve the network inference using a constraint least-squares approach named motif-guided network component analysis (mNCA). However, connection matrix usually contains a considerable amount of false positives and false negatives that make inference results questionable. To circumvent this problem, we propose a knowledge based stability analysis (kSA) approach to test the conditional relevance of individual TFs, by checking the discrepancy of multiple estimations of transcription factor activity with respect to different perturbations on the connections. The rationale behind stability analysis is that the consistency of observed gene expression and true network connection shall remain stable after small perturbations are applied to initial connection matrix. With condition-specific TFs prioritized by kSA, we further propose to use multivariate regression to highlight condition-specific target genes. Through simulation studies comparing with several competing methods, we show that the proposed schemes are more sensitive to detect relevant TFs and target genes for network inference purpose. Experimentally, we have applied stability analysis to yeast cell cycle experiment and further to a series of anti-estrogen breast cancer studies. In both experiments not only biologically relevant regulators are highlighted, the condition-specific transcriptional regulatory networks are also constructed, which could provide further insights into the corresponding cellular mechanisms.
(b) if only single TF's protein-DNA information is available:
this happens when protein-DNA binding relationship of individual TF is measured through experiments. Since original mNCA requires a complete connection matrix to perform estimation, an incomplete knowledge of single TF is not applicable for such approach. Moreover, binding information derived from experiments could still be inconsistent with gene expression levels. To overcome these limitations, we propose a linear extraction scheme called regulatory component analysis (RCA), which can infer underlying regulation relationships, even with partial biological knowledge. Numerical simulations show significant improvement of RCA over other traditional methods to identify target genes, not only in low signal-to-noise-ratio situations and but also when the given biological knowledge is incomplete and inconsistent to data. Furthermore, biological experiments on Escherichia coli regulatory network inferences are performed to fairly compare traditional methods, where the effectiveness and superior performance of RCA are confirmed.
The second part of the dissertation moves from protein-DNA interaction network up to protein-protein interaction network, to identify dys-regulated protein sub-networks by integrating gene expression data and protein-protein interaction information. Specifically, we propose a statistically principled method, namely Metropolis random walk on graph (MRWOG), to highlight condition-specific PPI sub-networks in a probabilistic way. The method is based on the Markov chain Monte Carlo (MCMC) theory to generate a series of samples that will eventually converge to some desired equilibrium distribution, and each sample indicates the selection of one particular sub-network during the process of Metropolis random walk. The central idea of MRWOG is built upon that the essentiality of one gene to be included in a sub-network depends on not only its expression but also its topological importance. Contrasted to most existing methods constructing sub-networks in a deterministic way and therefore lacking relevance score for each protein, MRWOG is capable of assessing the importance of each individual protein node in a global way, not only reflecting its individual association with clinical outcome but also indicating its topological role (hub, bridge) to connect other important proteins. Moreover, each protein node is associated with a sampling frequency score, which enables the statistical justification of each individual node and flexible scaling of sub-network results. Based on MRWOG approach, we further propose two strategies: one is bootstrapping used for assessing statistical confidence of detected sub-networks; the other is graphic division to separate a large sub-network to several smaller sub-networks for facilitating interpretations. MRWOG is easy to use with only two parameters need to be adjusted, one is beta value for performing random walk and another is Quantile level for calculating truncated posteriori mean. Through extensive simulations, we show that the proposed scheme is not sensitive to these two parameters in a relatively wide range. We also compare MRWOG with deterministic approaches for identifying sub-network and prioritizing topologically important proteins, in both cases MRWG outperforms existing methods in terms of both precision and recall. By utilizing MRWOG generated node/edge sampling frequency, which is actually posteriori mean of corresponding protein node/interaction edge, we illustrate that condition-specific nodes/interactions can be better prioritized than the schemes based on scores of individual node/interaction. Experimentally, we have applied MRWOG to study yeast knockout experiment for galactose utilization pathways to reveal important components of corresponding biological functions; we also applied MRWSOG to study breast cancer patient prognostics problems, where the sub-network analysis could lead to an understanding of the molecular mechanisms of antiestrogen resistance in breast cancer.
Finally, we conclude this dissertation with a summary of the original contributions, and the future work for deepening the theoretical justification of the proposed methods and broadening their potential biological applications such as cancer studies. / Ph. D.
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Crystal Structure Of Mycobacterium Tuberculosis Histone Like Protein HU And Structure Based Design Of Molecules To Inhibit MtbHU-DNA Interaction : Leads For A New Target. Structure Aided Computational Analysis Of Metal Coordinated Complexes Containing Amino Acids And Organic Moieties Designed For Photo Induced DNA CleavageBhowmick, Tuhin 04 1900 (has links) (PDF)
In bacteria, nucleoid associated proteins (NAPs) represent a prominent group of global regulators that perform the tasks of genome compaction, establishing chromosomal architecture and regulation of various DNA transactions like replication, transcription, recombination and repair. HU, a basic histone like protein, is one of the most important NAPs in Eubacteria. Mycobacterium tuberculosis produces a homodimeric HU (MtbHU), which interacts with DNA non-specifically through minor groove binding. Exploration for essential genes in Mtb (H37Rv) through transposon insertion has identified HU coding gene [Rv2986c, hupB; Gene Id: 15610123; Swiss-Prot ID: P95109)] to be vital for the survival and growth of this pathogen.
MtbHU contains two domains, the N-terminal domain which is considerably conserved among the HU proteins of the prokaryotic world, and a C–terminal domain consisting of Lys-Ala rich multiple repeat degenerate motifs. Sequence analysis carried out by the thesis candidate showed that MtbHU exhibits 86 to 100 percent identity within the N-term region among all the mycobacterium species and some of the members of actinobacteria, including important pathogens like M. tuberculosis, M. leprae, M. ulcerans, M. bovis, Nocardia; while C term repeat region varies relatively more. This strikingly high cross species identity establishes the MtbHU N-terminal domain (MtbHUN) as an important representative structural model for the above mentioned group of pathogens.
The thesis candidate has solved the X-ray crystal structure of MtbHUN, crystallized in two different forms, P2 and P21. The crystal structures in combination with computational analyses elucidate the structural details of MtbHU interaction with DNA. Moreover, the similar mode of self assembly of MtbHUN observed in two different crystal forms reveals that the same DNA binding interface of the protein can also be utilized to form higher order oligomers, that HU is known to form at higher concentrations. Though the bifunctional interface involved in both DNA binding and self assembly is not akin to a typical enzyme active site, the structural analysis identified key interacting residues involved in macromolecular interactions, allowing us to develop a rationale for inhibitor design. Further, the candidate has performed virtual screening against a vast library of compounds, and design of small molecules to target MtbHU and disrupt its binding to DNA. Various biochemical, mutational and biological studies were performed in the laboratory of our collaborator Prof. V. Nagaraja, MCBL, IISc., to investigate these aspects. After a series of iterations including design, synthesis and validation, we have identified novel candidate molecules, which bind to MtbHU, disrupt chromosomal architecture and arrest M. tuberculosis growth. Thus, the study suggests that, these molecules can serve as leads for a new class of DNA-interaction inhibitors and HU as a druggable target, more so because HU is essential to Mtb, but absent in human. Our study proposes that, targeting the nucleoid associated protein HU in Mtb can strategize design of new anti-mycobacterial therapeutics. Perturbation of MtbHU-DNA binding through the identified compounds provides the first instance of medium to small molecular inhibitors of NAP, and augurs well for the development of chemical probe(s) to perturb HU functions, and can be used as a fundamental chemical tool for the system level studies of HU-interactome.
Section I: “Crystal structure of Mycobacterium tuberculosis histone like protein HU and structure based design of molecules to inhibit MtbHU-DNA interaction: Leads for a new target.” of this thesis presents an elaborate elucidation of the above mentioned work.
The candidate has additionally carried out structure based computational and theoretical work to elucidate the interaction of amino acid based metal complexes which efficiently bind to DNA via minor-groove, major-groove or base intercalation interaction and display DNA cleavage activity on photo-irradiation. This understanding is crucial for the design of molecules towards Photodynamic Therapy (PDT). PDT is an emerging method of non-invasive treatment of cancer in which drugs like Photofrin show localized toxicity on photoactivation at the tumor cells leaving the healthy cells unaffected.
The work carried out in our group in close collaboration with Prof. A.R. Chakravarty of Inorganic and Physical Chemistry Department elaborates the structure based design of Amino acid complexes containing single Cu (II), such as [Cu(L-trp)(dpq)(H2O)]+ , [Cu (L-arg) 2](NO3)2 , Amino acid complexes containing oxobridged diiron Fe(III), such as [{Fe(L-his)(bpy)}2(μ-O)](ClO4)2 , [{Fe(L-his)(phen)}2(μ-O)](ClO4)2 , and Complexes containing Binuclear Cu(II) coordinated organic moiety, such as [{(dpq) CuII}2(μ-dtdp)2], which bind to DNA through minor groove/major groove/base intercalation interactions. Docking analysis was performed with the X-ray crystallographic structure of DNA as receptor and the metal complexes as ligands, to study the mode of binding to DNA and to understand the possible mode of DNA cleavage (single/double strand) when activated with laser.
Section II: “Structure based computational and theoretical analysis of metal coordinated complexes containing amino acids and organic moieties designed for photo induced DNA cleavage” of this thesis presents a detailed presentation of the above mentioned work.
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Interação entre o fator de transcrição CG9571 e módulos reguladores do gene pair-rule even-skipped da cascata de segmentação de Drosophila / Interaction between the transcription factor CG9571 and cis-regulatory modules of the pair-rule gene even-skipped of Drosophila segmentation cascadeGueller, Geison Castro da Silveira 22 May 2019 (has links)
O desenvolvimento do padrão de segmentação de Drosophila é estabelecido por uma cascata de genes de segmentação zigóticos. Estes genes são divididos em três classes (gap, pair-rule e segment-polarity) e codificam para fatores de transcrição (FT) que se ligam a módulos cis-reguladores (CRMs), reprimindo ou ativando genes alvo. A faixa 2 do gene pair-rule even-skipped (eve 2) é ativada pelos fatores maternos Bicoid e Hunchback e reprimida pelas proteínas gap Giant (Gt) e Krüppel. Estudos posteriores mostraram que o FT Sloppy-paired 1 (Slp 1) e provavelmente um outro FT forkhead também atuam na repressão de eve 2. O gene anotado CG9571 foi isolado em uma varredura como proteína forkhead candidata a repressão de eve 2. Estudos genéticos confirmaram essa possibilidade e revelaram que eve 1 também pode ser alvo deste FT. Este trabalho teve como objetivo verificar a interação de CG9571 com os CRMs eve 1 e eve 2. Para tanto, planejamos obter o domínio de ligação da proteína (CG9571 BD) e da proteína completa (CG9571 FL) e testar suas interações in vitro com fragmentos dos CRMs por meio da técnica de retardo da mobilidade eletroforética (EMSA). Obtivemos a quantidade necessária de DNA para os experimentos através de PCR e preparações plasmidiais de versões clonadas destes CRMs que já dispúnhamos em laboratório. Realizamos tentativas de obtenção de CG9571 BD por transcrição e tradução in vitro, mas esta estratégia não foi bem-sucedida e adotamos a estratégia de clonagem em vetor para expressão em células competentes bacterianas. O fragmento de CG9571 BD foi clonado com sucesso, mas não conseguimos verificar a expressão do polipeptídeo em duas linhagens de E. coli. Alteramos novamente nossa estratégia e clonamos o fragmento correspondente a CG9571 FL em vetor de expressão e conseguimos induzir sua expressão em bactéria, embora não tenha sido obter a proteína recombinante em forma solúvel. Prosseguimos para tentativas de recuperação da proteína a partir de corpos de inclusão. Foram empregados diferentes métodos para solubilização, renovelamento e purificação da proteína. Extratos da fração insolúvel solubilizada em diferentes concentrações de ureia foram submetidos a tentativas de purificação e renaturação por cromatografia de afinidade, mas não houve adsorção significativa de CG9571 FL em colunas com Ni2+ imobilizado. Preparações não puras contendo CG9571 FL foram obtidas através de procedimentos de renaturação destes extratos e foram utilizadas em ensaios de interação com os CRMs. Não houve detecção de retardo da mobilidade eletroforética dos fragmentos em gel. Foram observados efeitos de redução da quantidade de DNA detectado com brometo de etídio nas interações, mas este efeito foi considerado produto da ação de possíveis nucleases contaminantes nas preparações após investigação. Preparações de CG9571 FL puras foram obtidas por purificações a partir de SDS-PAGE, mas a maioria das interações da proteína solúvel com eve 1 e eve 2 não indicou formação de complexo. Obtivemos um único resultado positivo para a interação entre CG9571 FL e eve 2. Por não ter sido reproduzido, consideramos o resultado inconclusivo e novos experimentos serão conduzidos para dar continuidade à investigação da hipótese do trabalho / The development of Drosophila segmentation pattern is established by a cascade of zygotic segmentation genes. The zygotic genes are grouped in three classes (gap, pair-rule and segment-polarity) and code for transcription factors (TF) that bind to cis-regulatory modules (CRMs) with activation or repression roles. The stripe 2 of the pair-rule gene even-skipped (eve 2) is activated by the maternal factors Bicoid and Hunchback and repressed by the gap proteins Giant and Krüppel. Later studies showed that Sloppy-paired 1 (Slp 1) and probably another forkhead transcription factor also act for eve 2 repression. The annotated gene CG9571 was isolated in a search for putative forkhead protein repressors of eve 2. Genetic studies confirmed this possibility and reveled that eve 1 could also be a target for this TF. The aim of this work was to verify the interaction of CG9571 with the CRMs eve 1 and eve 2. To reach this aim, we planned to obtain the binding domain of the protein (CG9571 BD) or of the full-length protein (CG9571 FL) and to test their in vitro interactions with the eve 1 and eve 2 fragments by the electrophoretic mobility shift assay (EMSA). We obtained the necessary amount of DNA for the tests by PCR and plasmidial preparations of cloned versions of these CRMs that we already had in our laboratory. We made attempts to obtain CG9571 BD by in vitro transcription and translation system, but this strategy did not work and we adopted the cloning strategy to obtain the protein expressed by bacterial competent cells. CG9571 BD was cloned successfully, but we were not able to detect the polypeptide expression in two E. coli strains. We then turned to the CG9571 FL protein that we cloned and succeed to express it in bacteria, although we were not able to obtain the soluble recombinant form. We proceed for attempts of protein recovering from inclusion bodies. Different methods for solubilization, refolding and purification of the proteins were used. Extracts of the insoluble fraction solubilized in solutions with different urea concentrations were used in attempts of refolding and purification by affinity chromatography, but there was not significant CG9571 FL adsorption on columns with Ni2+ immobilized. We obtained impure preparations with CG9571 FL through procedures of refolding of these extracts and employed them on binding assays with the CRMs, but there was no gel shift detection. We observed reduction of the amount of DNA present in the binding reaction samples detected by ethidium bromide, but after further investigations this effect was considered the product of contaminant nuclease reaction from bacteria. Pure preparations of CG9571 FL were obtained by purification from SDS-PAGE, but there was no indication of complex formation on the most binding reaction assays with eve 1 and eve 2. We obtained only one positive result for the interaction between CG9571 FL and eve 2. However, the result was considered inconclusive because we were not able to reproduce it and new experiments will be conducted to investigate the hypothesis of this work
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