毕赤酵母X-33OCH1基因敲除菌株的构建及其表达低糖基化蛋白GM-CSF

Pichia pastoris X-33 with OCH1 gene deletion and its expression of glycoprotein GM-CSF

摘要:

摘要: 【目的】酵母表达外源糖蛋白时会对蛋白进行过度N-糖基化修饰，产生高甘露糖型糖链，影响蛋白的活性，其中α-1,6-甘露糖转移酶(och1p)在这一过程中起着关键作用。通过敲除毕赤酵母X-33的α-1,6甘露糖转移酶(och1p)基因，获得一个对糖蛋白进行低糖基化修饰的毕赤酵母表达系统。【方法】采用双交换同源重组敲除目的基因的方法，首先敲除赤酵母X-33的URA3基因，获得一个尿嘧啶营养缺陷型的X-33(ura3-)菌株；然后用URA3基因作为选择标记，敲除X-33(ura3-)的α-1,6甘露糖转移酶(och1p)基因，获得OCH1基因敲除的X-33(och1-)菌株。用X-33 (och1-)菌表达糖蛋白GM-CSF，分析GM-CSF蛋白糖链的变化。【结果】首次成功敲除了X-33的URA3和OCH1基因，与野生型相比，X-33(och1-)菌表达的GM-CSF蛋白过度糖基化修饰程度明显降低。【结论】X-33(och1-)菌株的构建提供了一个对蛋白低N-糖基化修饰的毕赤酵母表达系统，也为进一步的糖基化改造提供了良好的基础。

Abstract:

Abstract: [Objective] Glycoproteins derived from yeast expression systems are usually hyperglycosylated and contain non human N-glycans of the high mannose type. α-1,6-mannosyltransfe rase(och1p) plays a key role in modifying glycoproteins with high mannose-type N-glycans. Therefore, we engineered a P. pastoris X-33 strain with OCH1 gene deletion. This strain was further used as a host for production of glycoproteins with smaller N-glycans. [Methods] First, we knockout the URA3 gene of P.pastoris X-33 which encoding orotidine-5’-phosphate decarboxylase by double homologous recombination. Then, we knockout OCH1 gene using URA3 gene as a selecting marker, and obtained X-33( och1-) strain. After that, this mutant strain was used to expression the glycoprotein granulocyte-macrophage colony-stimulating factor (GM-CSF). [Results] Different from hyperglycosylated GM-CSF expressed in wild type P.pastori X-33, the glycoprotein expressed in X-33(och1-), containing smaller N-glycan. [Conclusion] The results suggested that X-33(och1-) strain can be used as an expression host for production of glycoproteins lacking the outer-chain hypermannoses, and a host could be used for further N-glycosylation engineering.