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Single-Copy Insertion of Split-GFP for the Restriction of Germline Expression in Caenorhabditis elegansAl Johani, Mohammed 11 1900 (has links)
Gene regulation in C. elegans germ cells depend on transgenerational chromatin modification and small RNA pathways. Germline silencing mechanisms evolved to repress foreign DNA from compromising the transfer of genetic information to progeny. Effective genetic tools that circumvent the silencing machinery will facilitate studies using this model organism. Specifically, translation of heat-shock inducible transgenes is inhibited in the germline making it challenging to transiently express enzymes to modify the genome. Here, we describe a genetic screen design that can be used to identify pathways that prevent germline expression of heat-shock induced transgenes. We use split-GFP (GFP1-10 and GFP11) to confine a genetic screen to germ cells. Stable transgenic lines with germline expression of single-copy integrated GFP11 were produced using MosSCI. The insertion lines will be used in RNAi or chemical mutagenesis screens for the germline de-repression of GFP1-10 expressed under heat-shock promoters. The screen is likely to identify candidate RNAi or chromatin factors involved in repressing heat-shock expression in the germline, particularly from extrachromosomal arrays. Inducible high-level expression in the germline from extrachromosomal arrays would be a valuable tool for large-scale genome engineering.
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Ingénierie d'un outil basé sur une GFP fragmentée pour l'étude des protéines multi-localisées chez les eucaryotes / Engineering a Split-GFP based tool to study multilocalized protein in EukaryotesBader, Gaëtan 15 December 2017 (has links)
Les aminoacyl-ARNt synthétases catalysent la formation des aminoacyl-ARNt, utilisés lors de la synthèse protéique et peuvent également former des complexes multi-synthétasiques (MSC). Chez S. cerevisiae, le complexe AME associe les glutamyl- et méthionyl-ARNt synthétases à la protéine d’ancrage Arc1 et joue un rôle primordial dans la coordination de l’expression des génomes nucléaire et mitochondrial. Tous les composants de ce MSC sont multi-localisés et assurent des fonctions essentielles dans d’autres compartiments. Pour étudier ces localisations multiples, nous avons élaboré un outil, basé sur la Split-GFP, qui nous permet de visualiser spécifiquement la fraction organellaire d’une protéine multi-localisée. Pour cela, la GFP a été séparée en deux fragments : i) β1-10, restreint à un compartiment subcellulaire et ii) β11, fusionné aux protéines d’intérêts. Cet outil nous a permis d’étudier diverses relocalisations, ainsi que de délimiter des signaux d’import. / Aminoacyl-tRNA synthetases catalyze aminoacyl-tRNA formation, required for protein synthesis but can also associate into multi-synthetase complexes (MSC). In S. cerevisiae, the AME complex contains glutamyl- and methionyl-tRNA synthetases bound to the anchor protein Arc1 and is responsible for the coordination of nuclear and mitochondrial genome expression. The three MSC partners are multi-localized and present simultaneously in several compartments. The detection of the organellar pools of these multilocalized proteins in vivo is difficult, since they are mainly cytosolic. Therefore, we engineered a split-GFP based localization tool that allows us to specifically visualize organellar fractions of multi-localized proteins. To do so, GFP was split into two parts: β1-10, restricted to a subcellular compartment and β11, fused to the protein of interest. This tool allowed us to study relocalization of cytosolic proteins and characterize targeting signals.
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Engineering Proteins with GFP: Study of Protein-Protein Interactions In vivo, Protein Expression and SolubilitySarkar, Mohosin M. January 2009 (has links)
No description available.
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Cell line and protein engineering tools for production and characterization of biologicsVolk, Anna-Luisa January 2017 (has links)
Our increasing understanding of disease mechanisms coupled with technological advances has facilitated the generation of pharmaceutical proteins, which are able to address yet unmet medical needs. Diseases that were fatal in the past can now be treated with novel biological medications improving and prolonging life for many patients. Pharmaceutical protein production is, however, a complex undertaking, which is by no means problem-free. The demand for more complex proteins and the realization of the importance of post-translational modifications have led to an increasing use of mammalian cells for protein expression. Despite improvements in design and production, the costs required for the development of pharmaceutical proteins still are far greater than those for conventional, small molecule drugs. To render such treatments affordable for healthcare suppliers and assist in the implementation of precision medicine, further progress is needed. In five papers this thesis describes strategies and methods that can help to advance the development and manufacturing of pharmaceutical proteins. Two platforms for antibody engineering have been developed and evaluated, one of which allows for efficient screening of antibody libraries whilst the second enables the straightforward generation of bispecific antibodies. Moreover, a method for epitope mapping has been devised and applied to map the therapeutic antibody eculizumab’s epitope on its target protein. In a second step it was shown how this epitope information can be used to stratify patients and, thus, contribute to the realization of precision medicine. The fourth project focuses on the cell line development process during pharmaceutical protein production. A platform is described combining split-GFP and fluorescence-activated droplet sorting, which allows for the efficient selection of highly secreting cells from a heterogeneous cell pool. In an accompanying study, the split-GFP probe was improved to enable shorter assay times and increased sensitivity, desirable characteristics for high-throughput screening of cell pools. In summary, this thesis provides tools to improve design, development and production of future pharmaceutical proteins and as a result, it makes a contribution to the goal of implementing precision medicine through the generation of more cost-effective biopharmaceuticals for well-characterized patient groups. / <p>QC 20170828</p>
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Etude de l'activation de la GTPase RhoB par complémentation split-GFP tripartite / Study of RhoB GTPase activation using tripartite split-GFP complementationKoraïchi, Faten 19 April 2016 (has links)
RhoB est une petite GTPase rapidement activée par les facteurs de croissance et les stress cellulaires, qui régule des processus biologiques fondamentaux comme la migration, l'angiogenèse, la réparation de l'ADN, l'apoptose ainsi que la réponse à des thérapeutiques anticancéreuses. L'activité des petites GTPases est finement régulée par leur localisation subcellulaire. Cependant, l'activation de RhoB en cellules vivantes n'avait jamais été investiguée. Ce travail a permis d'adapter et de valider une méthode innovante d'analyse des interactions protéine-protéine par complémentation split-GFP tripartite, pour la détection sensible et spécifique de l'activation des petites GTPases en cellules vivantes. Nous avons ensuite développé un modèle cellulaire optimisé par la combinaison de la technologie split-GFP tripartite et d'un intracorps anti-GFP amplificateur de fluorescence, pour détecter la régulation de l'activation de RhoB avec une haute résolution spatiale. Ce biosenseur a mis en évidence la translocation de la forme active de RhoB en réponse au sérum à partir des endosomes pour s'accumuler au niveau de la membrane plasmique, révélant ainsi une nouvelle plateforme de signalisation membranaire de RhoB. Ce biosenseur permettra d'analyser le profil d'activation de RhoB et d'autres petites GTPases, sous d'autres stimulations ou dans différents contextes cellulaires, et d'identifier leurs partenaires et les modulateurs de leur activation. / RhoB is a small GTPase that is rapidly activated in response to growth factors and cellular stress. It regulates fundamental biological processes such as cell migration, angiogenesis, DNA repair, apoptosis and response to anticancer therapies. Small GTPases activity is tightly regulated by their subcellular localization. However, RhoB activation had never been investigated in living cells. In this work, we have adapted and validated an innovative method of protein-protein interactions analysis using tripartite split-GFP complementation, for the sensitive and specific detection of small GTPases activation in living cells. Then, we developed an optimized cellular model by combining the tripartite split-GFP technology with an anti-GFP intrabody fluorescence-enhancer to detect the regulation of RhoB activation with high spatial resolution. This biosensor highlighted the translocation of active RhoB from endosomes to accumulate at the plasma membrane upon serum stimulation, revealing a novel membrane signaling platform of RhoB. Future studies based on this biosensor will enable the analysis of RhoB activation profile and other small GTPases upon various stimuli or in different cellular contexts, as well as the identification of the GTPases partners and activation modulators.
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Engineering membrane proteins for production and topologyToddo, Stephen January 2015 (has links)
The genomes of diverse organisms are predicted to contain 20 – 30% membrane protein encoding genes and more than half of all therapeutics target membrane proteins. However, only 2% of crystal structures deposited in the protein data bank represent integral membrane proteins. This reflects the difficulties in studying them using standard biochemical and crystallographic methods. The first problem frequently encountered when investigating membrane proteins is their low natural abundance, which is insufficient for biochemical and structural studies. The aim of my thesis was to provide a simple method to improve the production of recombinant proteins. One of the most commonly used methods to increase protein yields is codon optimization of the entire coding sequence. However, our data show that subtle synonymous codon substitutions in the 5’ region can be more efficient. This is consistent with the view that protein yields under normal conditions are more dependent on translation initiation than elongation. mRNA secondary structures around the 5’ region are in large part responsible for this effect although rare codons, as well as other factors, also contribute. We developed a PCR based method to optimize the 5’ region for increased protein production in Escherichia coli. For those proteins produced in sufficient quantities several additional hurdles remain before high quality crystals can be obtained. A second aim of my thesis work was to provide a simple method for topology mapping membrane proteins. A topology map provides information about the orientation of transmembrane regions and the location of protein domains in relation to the membrane, which can give information on structure-function relationships. To this end we explored the split-GFP system in which GFP is split between the 10th and 11th β-strands. This results in one large and one small fragment, both of which are non-fluorescent but can re-anneal and regain fluorescence if localized to the same cellular compartment. Fusing the 11th β-strand to the termini of a protein of interest and expressing it, followed by expression of the detector fragment in the cytosol, allows determination of the topology of inner membrane proteins. Using this strategy the topology of three model proteins was correctly determined. We believe that this system could be used to predict the topology of a large number of additional proteins, especially single-spanning inner membrane proteins in E. coli. The methods for efficient protein production and topology mapping engineered during my thesis work are simple and cost-efficient and may be very valuable in future studies of membrane proteins. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>
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