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KMID : 1094719980030010040
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Biotechnology and Bioprocess Engineering
1998 Volume.3 No. 1 p.40 ~ p.43
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Analysis of erythropoietin glycoform produced by recombinant CHO cells using the Lectin-blotting technique
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Chang Kern-Hee
Kim Kyung-Soo
Kim Jung-Hoe
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Abstract
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The glycosylation pattern of Erythropoietin (EPO), produced by recombinant CHO cells, was studied using the simple and rapid technique of ‘Lectin-blotting’. In this experiment we used three different kinds of lectins, MAA (Maackia amurensis agglutinine), RCA (Ricinus communis agglutinine), and DSA (Datura stramonium agglutinine), which bind to the terminal sialic acid, galactose, and the N-acetyllactosamine chain respectively. The lectin-blotting technique was used to analyze the carbohydrate structure of EPO produced in the presence of two physiologically active chemical compounds, ammonium and chloroquine. The effect of the ammonium ion on the glycosylation of EPO was studied because it accumulated in the medium mainly as a by-product of glutamine metabolism. Ammonium chloride significantly inhibited the sialylation of the terminal galactose residue at concentrations of 8 mM or more. Chloroquine, a potent inhibitor of glycosylation, inhibited terminal sialylation at concentrations of 100 and 200 μM, and at a concentration of 300 μM also inhibited N-acetyllactosamine chain synthesis.
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KEYWORD
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Erythropoietin, CHO, carbohydrate, glycoform, lectin
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