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. 2008 Feb 22;105(9):3232–3237. doi: 10.1073/pnas.0710412105

Fig. 1.

Fig. 1.

Engineering of a mucin-type glycosylation pathway in yeast. (a) Introduction of a mucin-type glycosylation pathway in yeast. UDP-Gal and UDP-GalNAc are synthesized from UDP-Glc and UDP-GlcNAc, respectively, by GalE protein in the cytosol. UGT2 protein transports UDP-Gal and UDP-GalNAc from the cytosol to the Golgi lumen. Next, ppGalNAc-Ts and core1 β1–3 GalT transfer GalNAc and Gal to polypeptides. (b) Construction of each gene integrated into the yeast genome. UGT2 protein was C-terminally 3× HA-tagged, and GalE protein had an N-terminal 3× myc tag. The glycosyltransferases were fused to the transmembrane domain of yeast MNN9, and a 3× FLAG-tag was inserted between the transmembrane domain and the catalytic domains of the transferases. MUC1a was fused to the α-factor prepro sequence for secretion into the medium, and a hexa-histidine tag was added to the C terminus.