Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells
Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells
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DOI：
10.1042/bj20031130
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发表时间：
2003-12-15
影响因子：
4.1
通讯作者：
Hansson, GC
中科院分区：
文献类型：
--
作者：
Bäckström, M;Link, T;Hansson, GC
我们已经开发了一种表达系统，用于在CHO-K1（中国仓鼠卵巢K1）细胞中大量生产重组MUC 1粘蛋白。人MUC 1的细胞外部分，包括16个MUC 1串联重复序列，作为与鼠IgG Fc的融合蛋白产生，具有用于去除Fc尾的插入肠激酶切割位点。产生稳定的产生MUC 1-IgG的CHO-K1克隆，并发现其将MUC 1-IgG分泌到培养基中。在生物反应器中的无蛋白质培养基中适应悬浮培养后，融合蛋白大量分泌（每天100 mg/l）到培养上清液中。从那里，MUC 1可以纯化到同质使用两步程序，包括肠激酶切割和离子交换色谱。从CHO-K1产生的MUC 1释放的寡糖的毛细管液相色谱MS鉴定出主要的O-聚糖为Galbeta 1 -3GaINAc（核心1）以及单唾液酸化和二唾液酸化核心1。聚糖平均占据串联重复序列中5个潜在O-糖基化位点中的4.3个，如通过部分去糖基化梭菌蛋白酶消化蛋白质的纳米液相色谱MS测定的。在乳腺癌细胞系T47 D中产生的MUC 1-IgG中发现了非常相似的O-聚糖谱和位点占用，其具有乳腺癌典型的O-糖基化。相比之下，在另一种乳腺癌细胞系MCF-7中产生的MUC 1-IgG显示出更复杂的模式，具有基于核心1和核心2的O-聚糖。这是第一次报道生产大量的重组MUC 1与乳腺癌样的O-糖基化，可用于乳腺癌的免疫治疗。
We have developed an expression system for the production of large quantities of recombinant MUC1 mucin in CHO-K1 (Chinese-hamster ovary K 1) cells. The extracellular part of human MUC1, including 16 MUC1 tandem repeats, was produced as a fusion protein with murine IgG Fc, with an intervening enterokinase cleavage site for the removal of the Fc tail. Stable MUC1-IgG-producing CHO-K1 clones were generated and were found to secrete MUC1-IgG into the culture medium. After adaptation to suspension culture in protein-free medium in a bioreactor, the fusion protein was secreted in large quantities (100 mg/l per day) into the culture supernatant. From there, MUC1 could be purified to homogeneity using a two-step procedure including enterokinase cleavage and ion-exchange chromatography. Capillary liquid chromatography MS of released oligosaccharides from CHO-K1-produced MUC1 identified the main O-glycans as Galbeta1-3GaINAc (core 1) and mono- and di-sialylated core 1. The glycans occupied on average 4.3 of the five potential O-glycosylation sites in the tandem repeats, as determined by nano-liquid chromatography MS of partially deglycosylated Clostripain-digested protein. A very similar O-glycan profile and site occupancy was found in MUC1-IgG produced in the breast carcinoma cell line T47D, which has O-glycosylation typical for breast cancer. In contrast, MUC1-IgG produced in another breast cancer cell line, MCF-7, showed a more complex pattern with both core 1- and core 2-based O-glycans. This is the first reported production of large quantities of recombinant MUC1 with a breast cancer-like O-glycosylation that could be used for the immunotherapy of breast cancer.