RECOMBINANT HUMAN INTERFERON-GAMMA - DIFFERENCES IN GLYCOSYLATION AND PROTEOLYTIC PROCESSING LEAD TO HETEROGENEITY IN BATCH CULTURE
RECOMBINANT HUMAN INTERFERON-GAMMA - DIFFERENCES IN GLYCOSYLATION AND PROTEOLYTIC PROCESSING LEAD TO HETEROGENEITY IN BATCH CULTURE
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DOI：
10.1042/bj2720333
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发表时间：
1990-12-01
影响因子：
4.1
通讯作者：
JENKINS, N
中科院分区：
文献类型：
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作者：
CURLING, EMA;HAYTER, PM;JENKINS, N
重组人干扰素-γ（Hu-IFN-γ）用免疫沉淀法和SDS/PAGE法分析了中国仓鼠卵巢细胞（CHO）产生的重组蛋白。该细胞系分泌多达12种分子量变体。三种变体回收后，酶去除所有N-连接的寡糖或糖基化被衣霉素抑制。三种多肽形式而不是单一形式的存在表明，蛋白水解裂解发生在糖基化和非糖基化形式的两个位点。蛋白水解切割的IFN-γ在细胞裂解物中比在分泌的糖蛋白中更普遍。与天然产生的IFN-γ一样，两者都是完全糖基化的IFN-γ。（天冬酰胺残基28和100被占据）和部分糖基化的产物（认为在位置Asn 28处被取代）被分泌。这是从糖基化产物的Mr和每种变体表达的唾液酸的相对量推断出来的。与天然产生的IFN-γ相反，非糖基化IFN-γ也由转染的CHO细胞分泌。当细胞在pH和溶解氧控制下在无血清培养基中分批培养时，非糖基化IFN-γ的比例增加。3小时后从3%增加到5%，达到总IFN-γ的30%。195 h后出现。当用代谢标记的IFN-γ时，未观察到所产生的糖基化蛋白比例的这种变化。用来自活跃生长的CHO细胞的无细胞上清液孵育96小时。这意味着细胞内糖基化发生改变，而不是分泌后寡糖侧链降解。IFN-γ的减少糖基化不依赖于培养基中的葡萄糖浓度，但可能与特异性生长和IFN-γ有关。生产率，因为这些在培养50小时后稳定下降，与非糖基化IFN-γ的增加的生产一致。
Recombinant human interferon-.gamma. (Hu-IFN-.gamma.) produced by Chinese-hamster ovary (CHO) cells was analysed by immunoprecipitation and SDS/PAGE. Up to twelve molecular-mass variants were secreted by this cell line. Three variants were recovered after enzymic removal of all N-linked oligosaccharides or when glycosylation was inhibited by tunicamycin. The presence of three polypeptide forms rather than a single form suggested that proteolytic cleavage had occurred at two sites in both the glycosylated and non-glycosylated forms. Proteolytically cleaved IFN-.gamma. was more prevalent in cell lysates than in the secreted glycoprotein. In common with naturally produced IFN-.gamma., both fully glycosylated IFN-.gamma. (asparagine residues 28 and 100 occupied) and partially glycosylated product (thought to be substituted at position Asn28) were secreted. This was deduced from the Mr of the glycosylated products and the relative amounts of sialic acid expressed by each variant. In contrast with naturally produced IFN-.gamma., non-glycosylated IFN-.gamma. was also secreted by the transfected CHO cells. When the cells were grown in batch culture in serum-free medium under pH and dissolved-oxygen control, the proportion of non-glycosylated IFN-.gamma. increased from 3 to 5% after 3 h, to 30% of the total IFN-.gamma. present after 195 h. This change in the proportion of glycosylated protein produced was not seen when metabolically labelled IFN-.gamma. was incubated for 96 h with cell-free supernatant from actively growing CHO cells. This implied that an alteration in intracellular glycosylation was occurring rather than a degradation of oligosaccharide side chains after secretion. The decrease in IFN-.gamma. glycosylation was independent of the glucose concentration in the culture medium, but could be related to specific growth and IFN-.gamma. production rates, as these declined steadily after 50 h of culture, in line with the increased production of non-glycosylated IFN-.gamma.