Construction of novel BHK-21 cell lines coexpressing Golgi resident or soluble forms of human alpha 2,6-sialyltransferase and alpha 1,3/4-fucosyltransferases together with secretory glycoproteins

The most frequently used mammalian host cell lines (BHK-21, CHO) for the production of recombinant therapeutic glycoproteins are incapable of producing alpha 2,6-sialylated or sialyl Lewis X containing oligosaccharide structures. Therefore, we have constructed novel stable BHK-21 cell lines by cotransfection of cells with plasmids encoding human secretory glycoproteins (beta-trace protein, erythropoietin, antithrombin III) and the membrane-bound or soluble forms of human glycosyltransferases alpha 2,6-sialyltransferase (ST6Gal), alpha 1,3-fucosyltransferase III (FT3) or VI (FT6). The secreted recombinant glycoproteins were purified from culture supernatants and were analyzed in detail using HPAE-PAD, mass spectrometry and NMR-analysis of their oligosaccharide chains. The soluble forms of the recombinant glycosyltransferases showed in vitro activity with oligosaccharides, glycolipids as well as glycoproteins. However, no modification of coexpressed secretory glycoproteins was detected. Thus a proper targeting/localization of the glycosyltransferases into the appropriate Golgi compartments is required for their in vivo activity on secreted recombinant glycoproteins. Such cell lines can be successfully used for the production of recombinant proteins with novel/tailored glycosylation characteristics that might alter and improve their biological in vivo properties (e.g. stability, tissue addressing).