The synthesis of proteins bearing unnatural amino acids has the potential to enhance and elucidate many processes in biochemistry and molecular biology. There are two primary methods for site specific unnatural amino acid incorporation, both of which use the cell’s native protein translating machinery: in vitro chemical acylation of suppressor tRNAs and the use of orthogonal amino acyl tRNA synthetases. Total chemical synthesis is theoretically possible, but current methods severely limit the maximum size of the product protein. In vivo orthogonal synthetase methods suffer from the high cost of the unnatural amino acid. In this thesis I sought to address this limitation by increasing cell density, first in shake flasks and then in a bioreactor in order to increase the yield of protein per amount of unnatural amino acid used. In a parallel project, I used the in vitro chemical acylation system to incorporate several unnatural amino acids, key among them the fluorophore BODIPYFL, with the aim of producing site specifically fluorescently labeled protein for single molecule FRET studies. I demonstrated successful incorporation of these amino acids into the trial protein GFP, although incorporation was not demonstrated in the final target, FEN1. This also served to confirm the effectiveness of a new procedure developed for chemical acylation.

Les glycosaminoglycanes (GAGs) sont des polymères de sucres linéaires, présents chez tous les animaux. Certaines bactéries pathogènes synthétisent également des polysaccharides identiques ou très similaires aux GAGs humains. Cette thèse a porté en particulier sur la synthèse de la chondroïtine sulfate et de l’héparosan qui font partie de cette famille de polysaccharides. L’intérêt pour ces deux GAGs est grandissant dans l’industrie pharmaceutique du fait des nombreuses applications médicales qu’ils pourraient permettre. La chondroïtine sulfate est d’ores et déjà extraite de tissus animaux ce qui peut engendrer des problèmes sanitaires, notamment des contaminations virales ou aux prions. En revanche, le procédé de production pour l’héparosan reste à mettre en place. Il est donc nécessaire de développer des procédés de production pour ces deux molécules. La synthèse enzymatique est une voie particulièrement prometteuse pour la production de la chondroïtine sulfate et de l’héparosan, et a fait l’objet de ce travail de thèse. / Glycosaminoglycans (GAGs) are long linear polysaccharide chains, found in all animals. Some pathogenic bacteria also synthesize polysaccharides identical or similar to human GAGs. This thesis deals with chondroitin sulfate and heparosan syntheses, members of the GAGs family. There is a growing interest in these two GAGs in the pharmaceutical industry due to numerous potential applications they offer. Chondroitin sulfate is currently extracted from animal tissues which can lead to sanitary problems such as viral or prion contaminations. On the other hand, a production process still needs to be developed for heparosan. Therefore, it is necessary to develop new methods for the production of these two polymers. Enzymatic synthesis, which is a promising alternative for the production of chondroitin sulfate and heparosan, was the subject of this thesis.

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