Murphy K P, Gagne P, Pazmany C, Moody M D
Endogen, Inc., Woburn, Massachusetts 01801-1059, USA.
Protein Expr Purif. 1998 Mar;12(2):208-14. doi: 10.1006/prep.1997.0832.
A Pichia pastoris expression clone has been developed to produce the human cytokine interleukin-17 (hIL-17). Characterization of purified recombinant hIL-17 made with this clone demonstrated that it shared many characteristics with hIL-17 produced in mammalian cells. The hIL-17 produced in Pichia had the correct N-terminus of natural mature hIL-17 and a glycosylation pattern similar to hIL-17 produced in mammalian cells; both Pichia and human cells add approximately 5 kDa of sugars via N-linked glycosylation and both express a mixture of the glycosylated and nonglycosylated forms. Gel filtration provides evidence that the Pichia produced hIL-17 exists as a dimer in solution. A combination of cation-exchange and gel-filtration chromatography yielded 3.5 mg of highly purified and biologically active hIL-17 from a 10-liter fermentation. These results show that P. pastoris is a useful system to produce recombinant hIL-17 in structure/function studies of this molecule.
已构建了一个毕赤酵母表达克隆来生产人细胞因子白细胞介素-17（hIL-17）。对用该克隆制备的纯化重组hIL-17的特性分析表明，它与在哺乳动物细胞中产生的hIL-17具有许多共同特征。在毕赤酵母中产生的hIL-17具有天然成熟hIL-17正确的N端，并且其糖基化模式与在哺乳动物细胞中产生的hIL-17相似；毕赤酵母和人类细胞都通过N-连接糖基化添加约5 kDa的糖，并都表达糖基化和非糖基化形式的混合物。凝胶过滤证明毕赤酵母产生的hIL-17在溶液中以二聚体形式存在。通过阳离子交换和凝胶过滤色谱相结合，从10升发酵液中获得了3.5 mg高度纯化且具有生物活性的hIL-17。这些结果表明，毕赤酵母是在该分子的结构/功能研究中生产重组hIL-17的有用系统。
