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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2013, Vol. 33 Issue (12): 79-85    
技术与方法     
利用N-糖基化修饰对β-甘露聚糖酶Man47的稳定性改造
谢春芳1,3, 黎玉凤2,3, 刘大岭1,3, 姚冬生2,3,4
1. 暨南大学生物工程学系 广州 510632;
2. 暨南大学生物医药研究院 广州 510632;
3. 广东省生物工程药物重点实验室 广州 510632;
4. 基因工程药物国家工程研究中心 广州 510632
The Stability Reconstruction of β-mannanase with N-glycosylation Modification
XIE Chun-fang1,3, LI Yu-feng2,3, LIU Da-ling1,3, YAO Dong-sheng2,3,4
1. Department of Bioengineering, Jinan University, Guangzhou 510632, China;
2. Institute of Biomedicine, Jinan University, Guangzhou 510632, China;
3. Guangdong Provincial Key Laboratory of Bioengineering Medicine, Guangzhou 510632, China;
4. National Engineering Research Center of Genetic Medicine, Guangzhou 510632, China
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摘要： N-糖基化是真核生物蛋白质最重要的翻译后修饰之一。以Armillariella tabescens β-甘露聚糖酶Man47为研究对象，利用计算化学对A. tabescens β-甘露聚糖酶Man47进行理性设计，经过分子对接、二级结构分析和糖基化的可行性分析后，构建具有EAS（enhanced aromatic sequence）序列的突变体g-123作为N-糖基化的突变位点。将其整合到毕赤酵母表达载体SMD1168上，通过电转化得到重组转化子。最后对突变体g-123的温度稳定性、酸碱稳定性、胃蛋白酶和胰蛋白酶抗性进行分析。结果表明：糖基化A. tabescens β-甘露聚糖酶Man47突变体g-123与野生型相比，其热稳定性、酸碱稳定性、蛋白酶抗性均得到不同程度的改善。
关键词： N-糖基化;  翻译后修饰;  蛋白质设计;  生物信息学    
Abstract: N-glycosylation is one of the most important posttranslational modification of proteins in eukaryotes. A rational strategy to introduce N-glycosylation site was proposed to Armillariella tabescens beta mannose Man47.Then g-123 mutant with EAS(enhanced aromatic sequence) sequence was built through the molecular docking, secondary structure analysis and feasibility analysis of glycosylation. The sequence of g-123 mutant was inserted into SMD1168 with the yeast α-mating factor, then transformed it into Pichia by electroporation to obtain the recombinants. Finally the thermal stability, acid and alkali stability, pepsin-resistance and trypsin-resistance of g-123 and wild type were analyzed. The results showed that compared with wild type, the thermal stability, acid and alkali stability, protease resistance of the mutant g-123 with glycosylation was improved.
Key words: N-glycosylation    Posttranslational modification    Protein design    Bioinformatics
收稿日期: 2013-09-03 出版日期: 2013-12-25
ZTFLH:  Q816  
基金资助: 国家“863”计划资助项目（2013AA102801）
通讯作者: 姚冬生，E-mail：tdsyao@jnu.edu.cn     E-mail: tdsyao@jnu.edu.cn
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引用本文:

谢春芳, 黎玉凤, 刘大岭, 姚冬生. 利用N-糖基化修饰对β-甘露聚糖酶Man47的稳定性改造[J]. 中国生物工程杂志, 2013, 33(12): 79-85.

XIE Chun-fang, LI Yu-feng, LIU Da-ling, YAO Dong-sheng. The Stability Reconstruction of β-mannanase with N-glycosylation Modification. China Biotechnology, 2013, 33(12): 79-85.

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https://manu60.magtech.com.cn/biotech/CN/    或    https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I12/79

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