构建并鉴定具有人类唾液酸化特征的稳定转染的 BHK-21 细胞系。
Construction and characterization of stably transfected BHK-21 cells with human-type sialylation characteristic.
机构信息
Department of Protein Glycosylation, GBF-Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, D-38124, Braunschweig, Germany.
出版信息
Cytotechnology. 1999 Jul;30(1-3):17-25. doi: 10.1023/A:1008049603947.
Abstract
The human Golgi enzyme CMP-NeuAc:Gal(beta1-4)GlcNAc-R alpha2,6-sialyltransferase (ST6N) was stably coexpressed with human erythropoietin (EPO) from a BHK-21A cell line. The cell line was characterized with respect to the expression and in vitro activity of the ST6N and the endogenous alpha2,3-sialyltransferase. Detailed structural analysis of the N-linked carbohydrates of the rhuEPO expressed from the new cell line was performed by HPAE-PAD-mapping, MALDI/TOF-MS and methylation analysis after purification of the recombinant protein by immunoaffinity chromatography. This is the first report describing that the human alpha2,6-sialyltransferase is capable of sialylating, apart from Gal(beta1-4)GlcNAc-R, also GalNAc(beta1-4)GlcNAc-R motifs in vivo, which is not the case for the endogenous BHK-cell alpha2,3-sialyltransferase.
摘要
人高尔基酶 CMP-NeuAc:Gal(beta1-4)GlcNAc-R alpha2,6-唾液酸转移酶 (ST6N) 与人红细胞生成素 (EPO) 一起从 BHK-21A 细胞系中稳定共表达。该细胞系的特征在于 ST6N 和内源性 alpha2,3-唾液酸转移酶的表达和体外活性。通过 HPAE-PAD 图谱、MALDI/TOF-MS 和免疫亲和层析纯化重组蛋白后的甲基化分析，对从新细胞系表达的 rhuEPO 的 N-连接糖进行了详细的结构分析。这是首次报道人类 alpha2,6-唾液酸转移酶除了 Gal(beta1-4)GlcNAc-R 之外，还能够在体内唾液酸化 GalNAc(beta1-4)GlcNAc-R 基序，而内源性 BHK 细胞 alpha2,3-唾液酸转移酶则不能。
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