N-糖基化位点突变的人IFN-λ1在毕赤酵母中的表达、纯化及表征

Expression, purification and characterization of N-glycosylation mutant human IFN-λ1 in Pichia pastoris

摘要:

IFN-λ1是Ⅲ型干扰素家族的一个成员，具有与Ⅰ型干扰素相似的功能。此前，我们已经从毕赤酵母表达获得了可溶性重组人干扰素-λ1。然而，毕赤酵母表达中的高糖基化带来了免疫原性，影响了蛋白质的生产纯化效率。为了克服这个缺点，文中构建了一种干扰素突变体 (rhIFN-λ1-Nm)，定点突变潜在糖基化位点。AOX1启动子与α因子信号序列存在的情况下，用甲醇诱导成功地实现了rhIFN-λ1-Nm在毕赤酵母GS115胞外分泌表达。对rhIFN-λ1-Nm进行纯化，获得了纯度>98%的产品，并对糖化水平、分子量、二级结构、N末端序列等理化性质和生物活性进行了研究。研究结果表明，rhIFN-λ1-Nm糖基化水平明显降低，蛋白质生产纯化收率显著提高，而对结构和生物活性无影响；糖基化位点突变rhIFN-λ1可以被开发为IFN-λ1的替代品，有望发展成为未来的生物免疫制剂。

关键词:人干扰素-λ1，N-糖基化突变，毕赤酵母

Abstract:

IFN-λ1 is a member of a new family of interferons called type Ⅲ IFNs with similar functions to type ⅠIFNs. Previously we obtained recombinant soluble human rhIFN-λ1 from Pichia pastoris. However, the hyper-glycosylation from P.?pastoris brings immunogenicity and low purification recovery rate. To overcome this disadvantage, in this study, we constructed an rhIFN-λ1 mutant (rhIFN-λ1-Nm) devoid of the potential N-glycosylation sites by site-directed mutagenesis. rhIFN-λ1-Nm was successfully expressed and secreted extracellularly in P. pastoris (GS115) using methanol inducible AOX1 promoter with α-mating factor signal sequence. rhIFN-λ1-Nm was purified and characterized. There was no significant difference between rhIFN-λ1-Nm and rhIFN-λ1 in structure and bioactivity. The molecular weight was low after N-glycosylation mutation whereas the glycosylation was much lower. The mutational rhIFN-λ1 (rhIFN-λ1-Nm) could legitimately be developed as substitutes for rhIFN-λ1, and thus it may be developed into a more promising therapeutic reagent in the future.

Key words:human IFN-λ1, N-glycosylation, Pichia pastoris