Minch S L, Kallio P T, Bailey J E
Department of Chemical Engineering, California Institute of Technology, Pasadena 91125, USA.
Biotechnol Prog. 1995 May-Jun;11(3):348-51. doi: 10.1021/bp00033a015.
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 beta-galactoside alpha(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 Sambacus nigra agglutinin (SNA)-biotin and streptavidin-R-phycoerythrin; SNA specifically binds to NeuAc alpha(2,6)Gal beta(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 NeuAc alpha(2,6)Gal beta(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.
对基因工程宿主细胞系中表达的一组寡糖生物合成酶进行基因改造，是一种可行的策略，可用于操纵在该细胞系中共表达的克隆糖蛋白上的寡糖。对于由工程化中国仓鼠卵巢（CHO）细胞系表达的重组人组织型纤溶酶原激活剂（tPA）的唾液酸化这一特定情况，该假设得到了验证。将大鼠肝脏β-半乳糖苷α(2,6)-唾液酸转移酶（2,6-ST）的基因克隆到载体pMSG中的MMTV启动子之后，并转染到表达tPA的CHO细胞系中。在使用黑接骨木凝集素（SNA）-生物素和链霉亲和素-R-藻红蛋白标记的细胞的流式细胞术分析中，筛选出的转染子显示出比对照明显更高的表面荧光；SNA特异性结合NeuAcα(2,6)Galβ(1,4)Glc-N-AcR连接，该连接由2,6-ST合成，在CHO细胞中通常不存在。对来自培养上清液的部分纯化的tPA进行SNA印迹分析表明，在2,6-ST转染子中合成的tPA具有末端NeuAcα(2,6)Galβ(1,4)Glc-N-AcR连接，而来自原始重组CHO细胞系的tPA则没有。除了可能使细胞培养中糖蛋白的糖基化更接近人类的糖基化之外，该策略的扩展还有可能调整克隆糖蛋白的药代动力学、靶向性和抗原特性。
