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Published May 1995 | public
Journal Article

Tissue Plasminogen Activator Coexpressed in Chinese Hamster Ovary Cells with α(2,6)-Sialyltransferase Contains NeuAcα(2,6)Galβ(1,4)Glc-N-AcR Linkages


Genetic alteration of the set of oligosaccharide biosynthesis enzymes expressed in a genetically engineered host cell line is a plausible strategy for manipulating the oligosaccharides on a cloned glycoprotein coexpressed in that cell line. This hypothesis was verified for the particular case of sialylation of recombinant human tissue plasminogen activator (tPA) expressed by an engineered Chinese hamster ovary (CHO) cell line. The gene for rat liver β-galactoside α(2,6)-sialyltransferase (2,6-ST) was cloned behind the MMTV promoter in the vector pMSG and transfected into a tPA-expressing CHO cell line. Selected and screened transfectants exhibited significantly greater surface fluorescence than controls in flow cytometric analyses of cells labeled with Sunabacus nigru agglutinin (SNA)-biotin and streptavidin-R-phycoerythrin; SNA specifically binds to NeuAcα(2,6)Gβ(1,4)Glc-N-AcR linkages, which are synthesized by 2,6-ST and which are not normally found on CHO cells. SNA blots of partially purified tPA from the culture supernatant demonstrated that tPA synthesized in the 2,6-ST transfectants possessed terminal NeuAcα(2,6)Gaβ(1,4)Glc-N-AcR linkages, while tPA from the original recombinant CHO cell line did not. Besides possibly allowing the production of glycoproteins in cell culture with glycosylation more closely resembling that in humans, extensions of this strategy have the potential to tailor the pharmacokinetics, targeting, and antigenic properties of cloned glycoproteins.

Additional Information

© 1995 American Chemical Society and American Institute of Chemical Engineers. Accepted December 20, 1994. Dr. James Paulson provided the 2,6-ST cDNA essential for this investigation. We thank Dr. Rochelle Diamond for conducting the flow cytometry analysis of the cells and for important advice. S.L.M. was supported by a National Science Foundation Graduate Research Fellowship.

Additional details

August 20, 2023
October 18, 2023