GLYCOSIDASE DIGESTION, ELECTROPHORESIS AND CHROMATOGRAPHIC ANALYSIS OF RECOMBINANT HUMAN GRANULOCYTE-COLONY-STIMULATING FACTOR GLYCOFORMS PRODUCED IN CHINESE-HAMSTER OVARY CELLS

被引：14

作者：

CLOGSTON, CL ^{[1
]}

HU, S ^{[1
]}

BOONE, TC ^{[1
]}

LU, HS ^{[1
]}

机构：

[1] AMGEN INC,AMGEN CTR,THOUSAND OAKS,CA 91320

来源：

JOURNAL OF CHROMATOGRAPHY | 1993年 / 637卷 / 01期

关键词：

D O I：

10.1016/0021-9673(93)83098-D

中图分类号：

O65 [分析化学];

学科分类号：

070302 ; 081704 ;

摘要：

Recombinant human granulocyte colony stimulating factor (G-CSF) produced in Chinese hamster ovary cells is glycosylated. The carbohydrate compositional analysis indicated that G-CSF molecule contains sialic acid, galactose and galactosamine. By isolation and characterization of the purified glycopeptides obtained from cleavages by Staphylococcal aureus V-8 protease and cyanogen bromide, the O-linked glycosylation site was confirmed to be a Thr residue at position 133. Neuraminidase and O-glycanase digestion followed by sodium dodecyl sulfate polyacrylamide and isoelectric focusing gel electrophoreses distinguished two possible carbohydrate structures attached at Thr-133: structure A, NeuNAc-Gal-beta(1,3)-GalNAc-O-Thr; and structure B, NeuNAc-Gal-beta(1,3)-[NeuNAc]-GalNAc-O-Thr. Different glycoforms, undigested or after glycosidase digestion, can also be separated by ion-exchange or reversed-phase high-performance liquid chromatography. The approach described in this report provides a simple and valuable procedure to characterize glycoprotein structures containing simple carbohydrate moieties.

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页码：55 / 62

页数：8

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