Wang Yuan, Jia Peng, Li Xuejun, Li Yuhong, Chen Peng
College of Life Sciences, Northwest A&F University, Yangling 712100, Shaanxi, China.
Horticulture College, Northwest A&F University, Yangling 712100, Shaanxi, China.
Sheng Wu Gong Cheng Xue Bao. 2018 Mar 25;34(3):421-428. doi: 10.13345/j.cjb.170254.
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.
N-聚糖酶是一类去糖基化酶，广泛应用于糖蛋白N-糖基化修饰的研究。本研究从水稻中克隆了一个GC含量较高（69.48%）的N-聚糖酶基因（OsPNGase A，XM_015775832），然后构建酵母分泌表达载体pPICZ(α)A-OsPNGase A，用于转化毕赤酵母。在毕赤酵母SMD1168H中诱导表达后，通过DEAE Sepharose和HisTrap HP层析法纯化目标蛋白，从1 L发酵培养基中获得12.3 mg OsPNGase A，比活性为258 U/mg。SDS-PAGE分析表明，纯化后的OsPNGase A为单一蛋白条带，与预期分子量一致。OsPNGase A可作用于水稻中重组表达的转铁蛋白、玉米中重组表达的抗生物素蛋白和辣根过氧化物酶。此外，OsPNGase A对抗生物素蛋白的活性高于市售的PNGase F。OsPNGase A在pH 6.0和40℃时消化活性最高，在中性和碱性环境中也具有活性。尽管OsPNGase A受到还原剂和表面活性剂的抑制，但在100 mmol/L β-ME或DTT中仍保持部分酶活性。因此，水稻OsPNGase A的成功表达为植物糖蛋白研究提供了新工具，酵母分泌表达体系的建立为PNGase A的制备奠定了基础。
