Construction and characterization of stably transfected BHK-21 cells with human-type sialylation characteristic

The human Golgi enzyme CMP-NeuAc:Gal(beta 1-4)GlcNAc-R alpha 2,6-sialyltran sferase (ST6N) was stably coexpressed with human erythropoietin (EPO) from a BHK-21A cell line. The cell line was characterized with respect to the ex pression and in vitro activity of the ST6N and the endogenous alpha 2,3-sia lyltransferase. Detailed structural analysis of the N- linked carbohydrates of the rhuEPO expressed from the new cell line was performed by HPAE-PAD-m apping, MALDI/TOF- MS and methylation analysis after purification of the re combinant protein by immunoaffinity chromatography. This is the first repor t describing that the human alpha 2,6-sialyltransferase is capable of sialy lating, apart from Gal(beta 1-4)GlcNAc-R, also GalNAc(beta 1-4) GlcNAc-R mo tifs in vivo, which is not the case for the endogenous BHK-cell alpha 2,3-s ialyltransferase.