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Développement d’une nouvelle méthodologie pour la production de molécules par ingénierie métabolique en délocalisant tout ou partie des réactions enzymatiques sur la surface de S. cerevisiae / Development of a new methodology for molecular production via metabolic engineering by relocating all or part of the enzymatic reaction on the surface of S. cerevisiaePauthenier, Cyrille 07 November 2016 (has links)
L’ingénierie métabolique est une discipline qui vise à modifier artificiellement le métabolisme d’un organisme afin de lui faire produire un composé chimique d’intérêt. L’une des problématiques fréquemment rencontrées pour la production de nouvelles molécules est l’impossible diffusion du produit formé dans le cytoplasme vers l’extérieur de la cellule. Ce phénomène engendre une accumulation de ce dernier à l’intérieur du micro-organisme qui limite sa capacité de production pour des raisons de cinétiques chimiques et de toxicité pour l’hôte. Enfin, cela complique fortement la récupération et la purification de la molécule d’intérêt, ce qui peut réduire à néant le rendement économique du procédé industriel.Durant cette thèse, nous avons exploré la possibilité de réaliser les dernières étapes de synthèse de composés imperméables pour la membrane à l’extérieur de la cellule en utilisant des enzymes accrochées à la surface de la levure par la technique de “yeast surface display”.Dans une première partie, nous avons regardé l’intérêt industriel de la bioéconomie, puis nous nous sommes intéressés aux problématiques liées à la perméabilité des membranes plasmiques. Dans un second temps, nous avons évalué les méthodes de mesures de la perméabilité des membranes biologiques et exploré la possibilité de développer une méthode prédictive en utilisant une technique de relation structure-propriété. Dans un troisième temps, nous avons évalué les systèmes de “yeast surface display” disponibles et cherché à en découvrir de nouveaux, adaptés à nos problématiques. Dans l'objectif de construire ces bibliothèques nous avons aussi réalisé un outils informatique, permettant de calculer de grands nombres de primers. Enfin, nous avons réalisé différents circuits de production de molécules modèles pour évaluer la pertinence de l’approche pour la production de composés imperméables. / Sustainable chemical production is one of the endeavour of the post-oil era. Amongst the possible techniques, metabolic engineering which aims at producing novel compounds through genetic engineering of micro-organism is seen as one of the most promising techniques. One of the problem met by metabolic engineers is often the absence of diffusion or pumping mechanism expelling the compound of interest produced in the cell cytoplasm towards the outer environment, which reduces the process efficiency because of kinetic and toxicity concerns.During this PhD, we explored the possibility of producing impermeable compounds on the surface of a cell by anchoring the last reaction enzyme using « Yeast surface display » techniques.As PhD disputation we first looked at the industrial interest of metabolic engineering in the whole bioeconomy framework. We then looked at the membrane permeability issues met for the production of some compounds. We evaluated the different membrane permeability techniques and explored the possibility realizing a predictive technique using quantitative structure-property relationship (QSAR). We evaluated the different yeast-display systems available and paved the way for the discovery of new systems more suitable for metabolic engineenering. We developped a dedicated program tool for large PCR fragment library design. Finally we built several toy metabolic pathways in yeast in order to evaluate the interest of the technique.
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Development of an AMP-SECreting Platform in E. coli for Simpler AMP Development (AMPSEC)Tomaro, Kyle 20 July 2022 (has links)
In the global fight against antibiotic resistance, the need for alternatives is more pressing than ever. Antimicrobial peptides (AMPs), short oligopeptides usually produced as part of the immune system of a host, have shown great promise against resistant bacteria, biofilms and even cancer cells. Engineering AMPs that are both specific to a set of bacteria and stable is among the main challenges of the field. Herein, we propose two separate tools to support these efforts.
The first one is an AMP SECretion system based in E. coli, dubbed AMPSEC, that can be used to produce active AMPs with specific targets (i.e., gram-positive bacteria or any specific specie). This recombinant protein system uses surface display technologies coupled with specific protease activity to express, export, and release functional AMPs that could readily affect neighbouring target bacteria. The AMPSEC would be ideal to screen AMP libraries, removing the need for purification or chemical synthesis in order to observe toxicity. It could also be used for AMP production, where the secreted AMPs would be purified from the growth medium by HPLC. Finally, if the recombinant system is inserted in a probiotic host, it might even be useful to deliver AMPs in the gut to treat dysbiosis. Herein, we explored six surface display apparatuses for their applicability for AMPSEC and found three out of the six being fully functional in transporting the cargo although the cleaving activity needs to be coordinated better with its localization at the outer membrane. A robust proof-of-concept workflow has also been established and used to evaluate the performance of those six display apparatuses.
The second is a bioinformatics approach to highlighting the relationship between the primary structure and the microbial target specificity of AMPs. Our method first clusters sequences from the DRAMP AMP repository using the Linclust algorithm. De novo motif discovery tools can then extract AMP sequence motifs relating to target specificity. These motifs could guide randomized sequence AMP library creation and decrease the number of inactive sequences generated. Clustering AMPs, however, proved to be rather challenging due to the large sequence length variation in the databases, the small sample size and their overall short lengths. It would then be necessary to design an algorithm suited to handle this specific kind of proteomics dataset. A library eventually created using the discovered motifs could then be used with AMPSEC. Combined, these two tools will further improve our ability to design stable AMPs targeting specific bacteria.
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Surface Displayed SNAP as a New Reporter in Synthetic BiologyScott, Felicia Yi Xia 10 July 2015 (has links)
The field of synthetic biology has leveraged engineering tools such as molecular cloning to create new biological components, networks, and processes. While many of these components and networks have been deployed in the cytosol, there is a shortage of systems that utilize the surface of the cell. In order to address this shortcoming, we have created a synthetic, surface-displayed substrate anchor for bacteria. This approach allows us to engineer surface-based synthetic biological systems as a complement to existing intracellular approaches. We leveraged the tools of synthetic biology to display a catalytically active enzyme that covalently bonds itself to benzylguanine (BG) groups. We created a fusion protein allowing us to place human O6-alkylguanine DNA alkyltransferase (hAGT), also known as SNAP, on the surface of a bacterial cell. Initially, we used this synthetic component as a tool for spatially segregating orthogonal synthetic gene outputs by visualizing an extracellular synthetic green fluorescent reporter, SNAP-Cell® 505-Star, simultaneously with an intracellular red fluorescent protein, mCherry. Moreover, we have shown that our construct enables cells to selectively bond to BG-conjugated magnetic beads. As a result, we have demonstrated that surface displayed SNAP facilitates engineering a direct channel between intracellular gene expression and extracellular material capture. In the near future, we believe this magnetic capture can be expanded as a sortable reporter for synthetic biology as a direct extension of this work. Moreover, our work serves as an enabling technology, paving the way for extracellular synthetic biological systems that may coexist orthogonally to intracellular processes. / Master of Science
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Controlled Hybrid Material Synthesis using Synthetic BiologyScott, Felicia Yi Xia 02 June 2017 (has links)
The concept of creating a hybrid material is motivated by the development of an improved product with acquired properties by amalgamation of components with specific desirable traits. These new attributes can range from improvements upon existing properties, such as strength and durability, to the acquisition of new abilities, such as magnetism and conductivity. Currently, the concept of an organic-inorganic hybrid material typically describes the integration of an inorganic polymer with organically derived proteins. By building on this idea and applying the advanced technologies available today, it is possible to combine living and nonliving components to synthesize functional materials possessing unique abilities of living cells such as self-healing, evolvability, and adaptability. Furthermore, artificial gene regulation, achievable through synthetic biology, allows for an additional dimension of the control of hybrid material function.
Here, I genetically engineer E. coli with a tightly controlled artificial protein construct, allowing for inducible expression of different amounts of the surface anchored protein by addition of varying concentrations of L-arabinose. The presence of the surface protein allows the cells to bind nonliving nanoparticle substrates, effectively turning the cells into living crosslinkers. By using the living crosslinker, I was able to successfully synthesize a robust, macroscale living-nonliving hybrid material with magnetic characteristics. Furthermore, by varying the particle size and inducer concentration, the resulting material exhibited alterations in structure and function. Finally, I was able to manipulate material kinetics within a PDMS channel by applying fluctuating magnetic fields and demonstrate material durability. These results demonstrate the ability to manipulate synthesis of living-nonliving hybrid materials, which demonstrate the potential for use in promising applications in areas such as environmental monitoring and micromachining. Additionally, this work serves as a foundational step toward the integration of synthetic biology with tissue engineering by exploiting the possibility of controlling material properties with genetic engineering. / Ph. D.
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Utilizing Solid Phase Cloning, Surface Display And Epitope Information for Antibody Generation and CharacterizationHu, Francis Jingxin January 2017 (has links)
Antibodies have become indispensable tools in diagnostics, research and as therapeutics. There are several strategies to generate monoclonal antibodies (mAbs) in order to avoid the drawbacks of polyclonal antibodies (pAbs) for therapeutic use. Moreover, the growing interest in precision medicine requires a well-characterized target and antibody to predict the responsiveness of a treatment. This thesis describes the use of epitope information and display technologies to generate and characterize antibodies. In Paper I, we evaluated if the epitope information of a well-characterized pAb could be used to generate mAbs with retained binding characteristics. In Paper II, the epitope on the complement protein C5 towards Eculizumab was mapped with surface display, the results of which explained the non-responsiveness of Eculizumab treatment among a patient group due to a mutated C5 gene. With this in mind, we showed efficacy in treatment of the mutated C5 variants using a drug binding to another site on C5, suggesting that our approach can be used to guide treatment in precision medicine. In Paper III, a Gram-positive bacterial display platform was evaluated to complement existing platforms for selection of human scFv libraries. When combined with phage display, a thorough library screening and isolation of nano-molar binders was possible. In Paper IV, a solid phase method for directed mutagenesis was developed to generate functional affinity maturation libraries by simultaneous targeting of all six CDRs. The method was also used to create numerous individual mutants to map the paratope of the parent scFv. The paratope information was used to create directed libraries and deep sequencing of the affinity maturation libraries confirmed the viability of the combination approach. Taken together, precise epitope/paratope information together with display technologies have the potential to generate attractive therapeutic antibodies and direct treatment in precision medicine. / <p>QC 20170418</p>
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Flödescytometrisk undersökning av inbindning mellan designade topdomänen från transferrinreceptorn till virala glykoproteiner för potentiell användning inom läkemedelsframtagning / Flow cytometric investigation of binding between the designed top domain of the transferrin receptor to viral glycoproteins for potential use in drug developmentRydell, Emma January 2022 (has links)
Machupovirus är ett virus som kan orsaka hemorragisk feber hos människor. Efter utvärdering av bindning mellan designade proteiner och virala glykoproteiner skulle proteinerna potentiellt kunna användas vid framtagning av ett proteinbasserat läkemedel mot hemorragisk feber. Syftet med studien var att efter riktad evolution och framrening av optimerade varianter av proteinet AP01 undersöka inbindningen till virala glykoproteiner mellan designade AP01 proteiner och transferrinreceptorn med hjälp av flödescytometrisk undersökning. Den fysiologiska nivån av järn i kroppen upprätthålls av transferrin (Tf) och transferrinreceptorn (TfR), ett transmembranprotein bestående av tre domäner. TfR apikala domän används av glykoprotein 1 (MGP1) och Plasmodium vivax för att ta sig in i celler genom receptormedierad endocytos. Med rekombinant genteknik kan rekombinanta plasmider skapas där en gen av intresse ligeras in i en plasmid med hjälp av DNA-ligas. I studien skapades rekombinanta plasmider pET29b+/AP01 S2.1, S2.2, S2.3, S3.3, S3.4 och S3.6 som transformerades till E. coli. Erhållna resultat från sekvensering visade att samtliga sex AP01-gener hade ligerats i vektorn men sekvensering av rekombinanta plasmider visade att endast pET29b+/AP01 S2.1, S2.2, S2.3 och S3.6 hade nukleotidsekvens utan mutationer. Proteinuttryck inducerades innan proteiner renades fram med immobilized metal ion affinity chromatography (IMAC). Den uppskattade molekylvikten hos de framrenade proteinerna var 18 kDa som bestämdes med sodium dodecyl sulfate – polyacrylamid gel electrophoresis (SDS-PAGE) vilket överrenstämde med den teoretiska molekylvikten. Flödescytometri användes för att undersöka inbindningsförmågan mellan de uttryckta proteinerna och glykoprotein 1 (MGP1). Interaktionsbindningen mellan de designade proteinerna och MGP1 är bättre än interaktionen mellan originalgen AP01 och MGP1. De designade proteinerna visar på en svag effekt i den utförda ”competition assay” som gjorts vilket kan förklaras med en ej optimal struktur hos de designade proteinerna eller närvaro av BSA. / Machupovirus is a virus that can cause hemorragic fever in humans. After evaluating the binding between designed proteins and viral glycoproteins, the proteins could potentially be used in the development of a protein-based drug for hemorrhagic fever. The aim of the study was to investigate the binding to viral glycoproteins between designed AP01 proteins and the transferrin receptor after directed evolution and purification of optimized variants of the AP01 protein by means of flow cytometric examination. The physiological level of iron in the body is maintained by transferrin (Tf) and the transferrin receptor (TfR), a transmembrane protein consisting of three domains. The apical domain of TfR is used by glycoprotein 1 (MGP1) and Plasmodium vivax to enter cells through receptor mediated endocytosis. With recombinant DNA technology, recombinant plasmids can be created where a gene of interest is ligated into a plasmid using DNA ligase. In this study, recombinant plasmids pET29b+/AP01 S2.1, S2.2, S2.3, S3.3, S3.4 and S3.6 were created and transformed into E. coli. Sequencing results showed that all six AP01 genes had been ligated into the vector but sequencing of recombinant plasmids showed that only endast pET29b+/AP01 S2.1, S2.2, S2.3 and S3.6 had nucleotid sequence without mutations. Protein expression was induced before proteins were purified by immobilized metal ion affinity chromatography (IMAC). The estimated molecular weight of the purified proteins was 18 kDa as determined by sodium dodecyl sulfate – polyacrylamid gel electrophoresis (SDS-PAGE) which was consistent with the theoretical molecular weight. Flow cytometry was used to examine the binding ability between the expressed proteins and glycoprotein 1 (MGP1). The interaction binding between the designed proteins and MGP1 is better than the interaction between the original gene AP01 and MGP1. The designed proteins show a weak effect in the “competition assay” preformed, wich can be explained by a non-optimal structure how the designed proteins or the presence of BSA.
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Staphylococcal surface display for protein engineering and characterizationLöfblom, John January 2007 (has links)
Even though our understanding of mechanisms such as protein folding and molecular recognition is relatively poor, antibodies and alternative affinity proteins with entirely novel functions are today generated in a routine manner. The reason for this success is an engineering approach generally known as directed evolution. Directed evolution has provided researchers with a tool for circumventing our limited knowledge and hence the possibility to create novel molecules that by no means could have been designed today. The approach is based on construction of high-complexity combinatorial libraries from which protein variants with desired properties can be selected. Engineered proteins are already indispensable tools in nearly all areas of life science and the recent advent of mainly monoclonal antibodies as therapeutic agents has directed even more attention to the field of combinatorial protein engineering. In this thesis, I present the underlying research efforts of six original papers. The overall objective of the studies has been to develop and investigate a new staphylococcal surface display method for protein engineering and protein characterization. The technology is based on display of recombinant proteins on surface of the Gram-positive bacteria Staphylococcus carnosus. In two initial studies, two key issues were addressed in order to improve the protein engineering method in regard to affinity discrimination ability and transformation efficiency. The successful results enabled investigation of the staphylococcal display system for de novo generation of affibody molecules from large combinatorial libraries. In this study, a high-complexity protein library was for the first time displayed on surface of Gram-positive bacteria and by means of fluorescence-activated cell sorting, specific affinity proteins for tumor necrosis factor-alpha were isolated. Moreover, in following papers, the staphylococcal display method was further improved and investigated for affinity determination, soluble protein production and epitope mapping purposes in order to facilitate downstream characterizations of generated affinity proteins. Taken together, in these studies we have demonstrated that the staphylococcal display system is a powerful alternative to existing technologies for protein engineering and protein characterization. / QC 20100809
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Engineering of Affibody molecules targeting the Alzheimer’s-related amyloid β peptideLindberg, Hanna January 2015 (has links)
<p>QC 20150922</p>
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Epitope mapping of antibodies towards human protein targetsHjelm, Barbara January 2011 (has links)
This thesis, based on five research papers, presents results from development and evaluation ofmethods for identifying the interaction site of antibodies on their antigens and the functional investigation of these in different assays. As antibodies have proven to be invaluable tools in diagnostics, therapy and basic research, the demand of characterizing these binding molecules has increased. Techniques for epitope mapping in a streamlined manner are therefore needed, particularly in high throughput projects as the Human Protein Atlas that aims to systematically generate two antibodies with separate epitopes towards all human proteins. In paper I we describe an approach to map the epitopes of polyclonal and monoclonal antibodies for the first time using staphylococcal display. This method was combined with peptide scanning and alanine scanning using suspension bead arrays, to create a streamlined approach of highresolution characterization of epitopes recognized by antibodies as demonstrated in paper II. Single epitopes were identified for the monoclonal antibodies and several (one to five) separate epitopes scattered throughout the antigen sequence were determined for each polyclonal antibody. Further, antibodies of different species origin showed overlapping binding epitopes. In paper III we studied the epitope patterns of polyclonal antibodies generated with the same antigen in different animals. Although common epitope regions could be identified the exact epitope pattern was not repeated, as some epitopes did not reoccur in the repeated immunizations. In paper IV, a potential biomarker for colon cancer, RBM3, was investigated using validated antibodies by epitope mapping and siRNA analysis. Finally, in paper V, a method for generating epitope-specific antibodies based on affinity purification of a polyclonal antibody is described. The generated antibodies were used in several immunoassays and showed a great difference in functionality. Paired antibodies with separate epitopes were successfully generated and could be used in a sandwich assay or to validate each other in immunohistochemistry. Taken together, in these studies we have demonstrated valuable concepts for the characterization of antibody epitopes. / QC 20120111
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Structural and Functional Analysis of Moraxella catarrhalis Adhesins MCAP and OMPCDAkimana, Christine 13 June 2007 (has links)
No description available.
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