| Autor: |
Bobrowicz, Piotr, Davidson, Robert C., Li, Huijuan, Potgieter, Thomas I., Nett, Juergen H., Hamilton, Stephen R., Stadheim, Terrance A., Miele, Robert G., Bobrowicz, Beata, Mitchell, Teresa, Rausch, Sebastian, Renfer, Eduard, Wildt, Stefan |
| Zdroj: |
Glycobiology; September 2004, Vol. 14 Issue: 9 p757-757, 1p |
| Abstrakt: |
A significant percentage of eukaryotic proteins contain posttranslational modifications, including glycosylation, which are required for biological function. However, the understanding of the structure–function relationships of N-glycans has lagged significantly due to the microheterogeneity of glycosylation in mammalian produced proteins. Recently we reported on the cellular engineering of yeast to replicate human N-glycosylation for the production of glycoproteins. Here we report the engineering of an artificial glycosylation pathway in Pichia pastoris blocked in dolichol oligosaccharide assembly. The PpALG3 gene encoding Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase was deleted in a strain that was previously engineered to produce hybrid GlcNAcMan5GlcNAc2 human N-glycans. Employing this approach, combined with the use of combinatorial genetic libraries, we engineered P. pastoris strains that synthesize complex GlcNAc2Man3GlcNAc2 N-glycans with striking homogeneity. Furthermore, through expression of a Golgi-localized fusion protein comprising UDP-glucose 4-epimerase and β-1,4-galactosyl transferase activities we demonstrate that this structure is a substrate for highly efficient in vivo galactose addition. Taken together, these data demonstrate that the artificial in vivo glycoengineering of yeast represents a major advance in the production of glycoproteins and will emerge as a practical tool to systematically elucidate the structure–function relationship of N-glycans. |
| Databáze: |
Supplemental Index |
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