Modular Assembly of Glycoproteins: Towards the Synthesis of GlyCAM-1 by Using Expressed Protein Ligation

摘要

Glycosylation is a vital protein modification for the normal growth and development of organisms. However, establishing the biological significance of specific covalently bound oligosaccharides is difficult owing to glycoprotein microheterogeneity, a phenomenon arising from the fact that protein glycosylation is not under direct genetic control. Consequently, a single glycoprotein can exist as a complex mixture of glycosylated forms termed glycoforms and analytical difficulties arising from this property have been a driving force for the development of new methods for the synthesis of glycoproteins with predefined oligosaccharides. Although bacteria lack the protein-glycosylation machinery of mammalian cells, they can be employed to express large quantities of recombinant proteins which allows subsequent chemical modifications to be monitored by using standard biophysical techniques. More recently it has been shown that non-native N-linked glycans can be assembled in Escherichia coli after transformation with the N-linked glycosylation apparatus from Campylobacter jejuni and raises the possibility of glycan engineering in bacteria. Furthermore, bacteria can produce useful protein fragments such as C-terminal thioesters which can be used in convergent protein-coupling techniques, such as native chemical ligation and expressed protein ligation (EPL). Glycoprotein assembly is particularly suited to EPL since synthetically derived glycopeptides can be fused with bacterially derived protein fragments of large molecular weight to give glycoproteins.