水稻N-糖酰胺酶基因 (OsPNGase A) 的克隆及在毕赤酵母中的分泌表达

Cloning of Oryza sativa N-glycanase gene (OsPNGase A) and its expression in Pichia pastoris

摘要:

N-聚糖酶是一类广泛应用于糖蛋白的N-糖基化修饰研究中的去糖基化酶。本研究通过RT-PCR从水稻中克隆了一个高GC含量 (69.48%) 的N-聚糖酶基因 (OsPNGase A, XM_015775832)，通过无缝克隆技术构建酵母分泌型表达载体pPICZ(α)A-OsPNGase A，在毕赤酵母SMD1168H中进行诱导表达，发酵液经DEAE Sepharose阴离子交换层析和HisTrap HP金属离子螯合层析纯化，产量可达到12.3 mg/L，比活力为258 U/mg。SDS-PAGE结果显示，纯化的OsPNGase A为单一条带且与预期分子量一致。OsPNGase A能作用于水稻中重组表达的人转铁蛋白 (TRF)、玉米中重组表达的鸡蛋抗生物素蛋白 (Avidin) 以及辣根过氧化物酶 (HRP)，并且对Avidin的酶切效果优于商业化的PNGase F。OsPNGase A反应的最适pH和温度分别为pH 6.0和40 ℃，在中性和碱性以及含有100 mmol/L还原剂β-ME和DTT的条件下仍具有活性。水稻OsPNGase A的成功表达为植物糖蛋白的研究提供一个新的工具酶，酵母分泌表达体系的建立为PNGase A的大量制备奠定了基础。

Abstract:

N-glycanase is a class of deglycosylation enzymes, widely used in the study of N-glycosylation modification of glycoprotein. In this study, an N-glycanase gene (OsPNGase A, XM_015775832) with high GC content (69.48%) was cloned from rice and then the yeast secretory expression vector pPICZ(α)A-OsPNGase A was constructed for the purpose of transformation to Pichia pastoris. After induction in Pichia pastoris SMD1168H, the target protein was purified by DEAE Sepharose and HisTrap HP chromatography, with a yield of 12.3 mg OsPNGase A from 1 L fermentation medium, showing a specific activity of 258 U/mg. SDS-PAGE revealed that the purified OsPNGase A was a single band and showed consistentcy with the expected molecular weight. OsPNGase A could act on transferrin recombinantly expressed in rice, avidin recombinantly expressed in corn and horseradish peroxidase. Furthermore, OsPNGase A showed higher activity than commercial PNGase F towards avidin. OsPNGase A displayed the highest digestion activity at pH 6.0 and 40 °C, and was also active in the neutral and alkaline environment. Despite the fact that OsPNGase A was inhibited by reducing agents and surfactants, it still maintained partial enzymatic activity in 100 mmol/L β-ME or DTT. Therefore, the successful expression of rice OsPNGase A provides a new tool for the study of plant glycoproteins and the establishment of yeast secretion expression system lays the foundation for the preparation of PNGase A.