Man5GlcNAc2哺乳动物甘露糖型糖蛋白的毕赤酵母表达系统构建

Construction of yeast Pichia pastoris to produce Man5GlcNAc2 mammalian mannose-type glycoprotein

摘要:

蛋白的糖基化对蛋白的活性、高级结构及功能都有重要的影响。酵母表达的糖蛋白不同于哺乳动物表达的杂合型或复杂型糖蛋白，而是高甘露糖型或过度甘露糖化糖蛋白。在前期成功敲除毕赤酵母α-1,6-甘露糖转移酶(Och1p)基因、阻断毕赤酵母过度糖基化，获得毕赤酵母过度糖基化缺陷菌株GJK01 (ura3、och1) 的基础上，通过表达不同物种来源的α-1,2-甘露糖苷酶I (MDSI) 的活性区与酵母自身定位信号的融合蛋白，并通过DSA-FACE (基于DNA 测序仪的荧光辅助糖电泳) 分析筛选报告蛋白HSA/GM-CSF (人血清白蛋白与粒细胞-巨噬细胞集落刺激因子融合蛋白) 的糖基结构，发现当编码酿酒酵母α-1,2-甘露糖苷酶 (MnsI) 基因的内质网定位信号与带有完整C-端催化区的拟南芥MDSI基因融合表达时，毕赤酵母工程菌株能够合成Man₅GlcNAc₂ 哺乳动物甘露糖型糖蛋白。这为在酵母体内合成类似于哺乳动物杂合型或复杂型糖基化修饰的糖蛋白奠定了基础。

Abstract:

Glycosylation is vital for activity, higher structure and function of protein. Glycoproteins derived from yeast contain N-glycan of high mannose type and are usually hyperglycosylated, while those from mammalian cells contain N-glycan of hybrid or complex type. We introduced the α-1,2-mannosidase I (MDSI) into yeast cells, which catalyzed an essential proceeding of N-glycan structures from Man₈GlcNAc₂ to Man₅GlcNAc₂. The plasmids contained MDSI genes from Homo sapiens [HMDSI(Δ185)] or Arabidopsis thaliana [ATMDSI(Δ48)], and three ER-signals were used to be transformed a mutant Pichia pastoris GJK01 , respectively. The reporter protein HSA/GM-CSF (human serum albumin and granulocyte-macrophage colony stimulating factor fusion protein) was expressed and its N-glycans were analyzed by DSA-FACE (DNA sequencer assisted fluorophore-assisted carbohydrate electrophoresis). The plasmid contained ER-ScMnsI-ATMDSI(Δ48) was expressed in Pichia pastoris, the Man₅GlcNAc₂ N-glycan on secreted glycoprotein HSA/GM-CSF was observed. The research reported here provided basic substrate to obtain the hybrid- and complex-type glycans in mammalian cell.