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Ingénierie de glycoside hydrolases pour la glycosylation des protéines recombinantes

Abstract : The control of glycosylation of recombinant proteins presents a considerable stake in the development of therapeutic proteins production in heterologous expression systems. Indeed glycosylation plays a key role in their properties, in particular pharmacokinetic properties. The remodelling of N-glycosylation of recombinant proteins was considered via the use of glycosynthase Cel7B E197A from Humicola insolens. This enzyme must first of all be modified in order to be able to accomodate an N-acetylglucosaminyl group in the acceptor subsite +1. Molecular modelling studies highlighted two amino acids which could prevent from positioning a carbohydrate unit substituted in position 2. Various mutants were prepared by site-directed mutagenesis in order to study their substrate specificity. Their glycosynthase activities were characterized, showing the influence of the introduced mutations. Even if the desired substrate specificity has not been obtained, a modification of the regioselectivity of the glycosynthase was highlighted with a substrate substituted in position 2 by an azido group.
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Contributor : Sophie Blanchard <>
Submitted on : Tuesday, January 25, 2005 - 6:29:13 PM
Last modification on : Friday, November 6, 2020 - 4:05:24 AM
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  • HAL Id : tel-00008252, version 1



Sophie Blanchard. Ingénierie de glycoside hydrolases pour la glycosylation des protéines recombinantes. Autre. Université Joseph-Fourier - Grenoble I, 2004. Français. ⟨tel-00008252⟩



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