Enabling bacterial production of glycoproteins by directed evolution of a glycosyl transferase

• Unité de recherche: INNOSUISSE
• Numéro de projet: 10094.1;5 PFLS-LS
• Titre du projet anglais: Enabling bacterial production of glycoproteins by directed evolution of a glycosyl transferase

Description succincte (Allemand)

Enabling bacterial production of glycoproteins by directed evolution of a glycosyl transferase.

Description succincte (Anglais)

Enabling bacterial production of glycoproteins by directed evolution of a glycosyl transferase

Résumé des résultats (Abstract) (Allemand)

Various oligosaccharides can be transferred from undecaprenylpyrophosphate donors to asparagine residues of acceptor proteins by the Campylobacter jejuni derived oligosaccharyltransferase PglB. Recently it was shown in Wacker et al. (2006), that only oligosaccharides with a C2¿ N-acetyl sugar at the reducing end can be transferred by PglB. When undecaprenylpyrophosphate activated oligosaccharides with a hexose at the reducing end were presented to the oligosaccharyltransferase, no glycosyslated product could be detected. However, many medically relevant oligosaccharide antigens such as the lipidAlinked antigen of S. typhimurium have hexoses at the reducing end of the repeating unit. It is known both from eucaryotoc and prokaryotic organism that hexoses can be linked to asparagine residues of proteins. It was also recently shown that oligosaccharyltransferases have a relaxed substrate specificity (Faridmoayer et al., 2008). Directed evolution is a powerful method for obtaining enzymes with improved properties (acceptance of novel substrates etc.). Within this project we attempt to evolve PglB such that it can transfer a hexose at the reducing end. This technology represents a breakthrough for the cost-efficient production of conjugated vaccines.

Résumé des résultats (Abstract) (Anglais)

Various oligosaccharides can be transferred from undecaprenylpyrophosphate donors to asparagine residues of acceptor proteins by the Campylobacter jejuni derived oligosaccharyltransferase PglB. Recently it was shown in Wacker et al. (2006), that only oligosaccharides with a C2¿ N-acetyl sugar at the reducing end can be transferred by PglB. When undecaprenylpyrophosphate activated oligosaccharides with a hexose at the reducing end were presented to the oligosaccharyltransferase, no glycosyslated product could be detected. However, many medically relevant oligosaccharide antigens such as the lipidAlinked antigen of S. typhimurium have hexoses at the reducing end of the repeating unit. It is known both from eucaryotoc and prokaryotic organism that hexoses can be linked to asparagine residues of proteins. It was also recently shown that oligosaccharyltransferases have a relaxed substrate specificity (Faridmoayer et al., 2008). Directed evolution is a powerful method for obtaining enzymes with improved properties (acceptance of novel substrates etc.). Within this project we attempt to evolve PglB such that it can transfer a hexose at the reducing end. This technology represents a breakthrough for the cost-efficient production of conjugated vaccines.