Expression of chondroitin-4-O-sulfotransferase in Escherichia coli and Pichia pastoris
Expression of chondroitin-4-O-sulfotransferase in Escherichia coli and Pichia pastoris
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DOI：
10.1007/s00253-017-8411-5
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发表时间：
2017-09-01
影响因子：
5
通讯作者：
Li, Ming
中科院分区：
文献类型：
--
作者：
He, Wenqin;Zhu, Yuanyuan;Li, Ming
硫酸软骨素是一种名为糖胺聚糖的线性硫酸盐多糖。它们是重要的营养食品和医药产品，通过软骨素磺基转移酶对未经硫酸盐化的软骨素或皮肤多糖前体的作用而生物合成。虽然参与硫酸软骨素生物合成的酶是众所周知的，但这些结合在膜上的高尔基体酶的克隆和表达仍然是一个挑战。人类主要的软骨素-4-磺基转移酶C4ST-1已在哺乳动物CHO、COS-7和HEK 293细胞中克隆和表达，其活性需要糖基化。在本研究中，我们设计了一个C4ST-1结构，并在大肠杆菌和巴斯德毕赤酵母中以非糖化和糖化形式表达。当作用于微生物制备的非硫酸软骨素底物时，两种构建物显示出相似的活性，尽管动力学参数不同。此外，糖基化形式的C4ST-1的稳定性低于非糖基化形式。
Chondroitin sulfates are linear sulfated polysaccharides called glycosaminoglycans. They are important nutraceutical and pharmaceutical products that are biosynthesized through the action of chondroitin sulfotransferases on either an unsulfated chondroitin or a dermatan polysaccharide precursor. While the enzymes involved in the biosynthesis of chondroitin sulfates are well known, the cloning end expression of these membrane-bound Golgi enzymes continue to pose challenges. The major chondroitin-4-sulfotransferase, Homo sapiens C4ST-1, had been previously cloned and expressed from mammalian CHO, COS-7, and HEK 293 cells, and its activity was shown to require glycosylation. In the current study, a C4ST-1 construct was designed and expressed in both Escherichia coli and Pichia pastoris in its non-glycosylated and glycosylated forms. Both constructs showed similar activity albeit different kinetic parameters when acting on a microbially prepared unsulfated chondroitin substrate. Moreover, the glycosylated form of C4ST-1 showed lower stability than the non-glycosylated form.