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Conception de protéines artificielles multidomaines

Abstract : The creation of new protein functions based on recognition and molecular assembly is not only a major goal in biotechnology but is also a means to understand the relation structure/function of proteins involved in interaction processes. Today, libraries of artificial proteins obtained by engineering can be a source of proteins with recognition properties similar to the properties of antibodies.The team Protein Engineering and Modeling has thus created a library of proteins with structural repeats called the “alphaReps”. The alphaReps present remarkable properties in terms of production and stability. Unlike most of the antibodies and their derivatives, they can even be expressed and functional in the cytoplasm of eukaryotic cells. Such objects can therefore be used as building bricks in modular engineering. The construction of new optimized recognition functions both in specificity and in affinity can then be possible by rearranging or duplicating these elementary bricks.The first part of this thesis project consisted in the construction and study of the biophysical properties of bidomain proteins based on alphaRep in order to have a better understanding of the behaviour of such constructions. Beside the fundamental aspect of this question, this study will give the “rules” to modulate the interactions between these proteins in a controlled way. The results show that it is possible to create new functions such as avidity, cooperativity, conformational change, simply by adding a linker between two alphaReps.In a second step, the goal was to develop, with the bidomain proteins previously studied, new biosensors based on the FRET (Förster Resonance Energy Transfer) which can be used in vivo and in vitro. This second part presents two biosensors with limits of detection in the nanomolar range. Since the alphaReps used in these constructions can be changed depending on the chosen target, it is a proof of concept which can be adapted to any desired target.Finally, the third part of this thesis focused on the development of genetically codable biosensors. These biosensors have the particular advantage of being usable directly after production and therefore no longer require a chemical coupling step. The results show that the development of such biosensors is worth considering but an optimization is still required in order to improve their specificity, their stability and their detection capacity.
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Submitted on : Friday, December 18, 2020 - 9:53:06 AM
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Corentin Léger. Conception de protéines artificielles multidomaines. Biologie structurale [q-bio.BM]. Université Paris Saclay (COmUE), 2018. Français. ⟨NNT : 2018SACLS384⟩. ⟨tel-03081249⟩



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