α-1,6-甘露糖转移酶基因敲除的毕赤酵母菌株构建及其用于融合蛋白HSA/GM-CSF 表达的研究

A Pichia pastoris with α-1,6-mannosyltransferases Deletion and its Use in Expression of HSA/GM-CSF Chimera

摘要:

酵母对蛋白的糖基化修饰过程不同于哺乳动物，其特点为产生高甘露糖型糖基且易发生过度糖基化。本研究通过两步基因重组敲除目标基因的方法成功敲除了毕赤酵母中的α-1,6-甘露糖转移酶（och1p）基因,获得了och1p敲除的菌株。以此为基础，构建了高效表达人血清白蛋白与粒细胞-巨噬细胞集落刺激因子融合蛋白（HSA/GM-CSF）的工程酵母，与野生型毕赤酵母表达的过度糖基化HSA/GM-CSF不同，och1p敲除菌表达的该融合蛋白糖基化程度明显降低，这为该融合蛋白的开发提供了重要基础。och1p敲除菌株的构建不仅提供了一个对糖蛋白进行低糖基化修饰的毕赤酵母表达系统，而且为进一步的酵母糖基工程改造提供了基础。

Abstract:

Yeast is a widely used host for recombinant protein expression. However, glycoproteins derived from yeast contain N-glycan of high mannose type and are usually hyperglycosylated. α-1,6-mannosyltransferases gene (och1) encodes the enzyme that initiates the first step of out-chain elongation of high mannose type N-glycan in yeast, which is different from that in human. So, a high efficient method to knockout target gene by two-step recombination was established and was used to delete och1. In the first recombinant, a plasmid with och1::ADE1 and ura3 gene was linearized in the downstream of och1 and inserted to the och1 site of P.pastoris genome, where the upstream and downstream of och1 were duplicated. In the second recombinant, the duplicated fragments of och1 were exchanged and the och1 deletion strains were selected on the plates containing 5-FOA, but no adenine. Then the och1 deletion strain was applied to express an human serum albumin (HSA) granulocyte-macrophage colony-stimulating factor (GM-CSF) chimera. Different with the hyperglycosylated HSA/GM-CSF chimera expressed in wild type P.pastoris, the chimera expressed in the och1 deletion strain, contained smaller N-glycan. The results suggested that the och1 mutant yeast may be more suitable for production of recombinant glycoproteins. And the och1 deletion strain could be used for further re-engineering to produce complex human glycoproteins.