Expression of Human Gal β1,3CalNAcα2,3-Sialytransferase (hST3GalⅡ ) in E. coli and Subcellular Localization of the Enzyme in Cells from Rat Liver Tissue

다국어 초록 (Multilingual Abstract)

GalB1,3 GalNAc α2,3-sialyltransferase(ST3Gal I and II) is the enzyme that transfers CMP-NeuAc with an α2,3-linkage to a galactose residue O-linked to a threonine/serine of a glycoprotein, and to the terminal galactose residues of glycolipids. Compared to ST3Gal I with the same acceptor substrate specificity, ST3Gal II exhibits much more acceptor substrate preference for glycolipids than for O-linked oligosaccharide of glycoproteins. For expression of human GalB-1,3GalNAc α2,3-sialyltransferase (hST3Gal II) in E.coli, the expression plasmid (pET-hST3Gal II) was constructed. The recombinant protein expressed by induction with IPTG was accumulated as inclusion bodies, which correspond to molecular mass of approximately 31-kDa on SDS-PAGE, in the insoluble fraction of cells. Treatment of inclusion bodies with Triton X-100 and EDTA yielded a high proportion of the recombinant hST3Gal II with a large increase in purity. To investigate the subcellular localization of ST3Gal II, polyclonal antibody to the recombinant hST3Gal II(rhST3Gal II) was produced. By immunoblot analysis using this antibody the 48-kDa protein which probably corresponds to ST3Gal II was detected in the Golgi fraction of rat liver, suggesting that ST3Gal II is localized in the Golgi apparatus.