生物学杂志

生物学杂志

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阿维链霉菌来源EndoH在枯草芽孢杆菌中的表达及应用

  

  1. 1.糖化学与生物技术教育部重点实验室， 无锡 214122；2.江南大学生物工程学院， 无锡 214122
  • 出版日期:2016-08-18 发布日期:2016-08-18
  • 通讯作者: 关锋，教授，主要从事糖生物学研究，E-mail：fengguan@jiangnan.edu.cn
  • 作者简介:梁标，硕士研究生，研究方向为分子生物学，E-mail：348317995@qq.com
  • 基金资助:
    自然科学基金项目（项目号：81201572）

The heterologous expression and application of Endo-β-N-acetyglucosaminidase H from Streptomyces avermitilis in Bacillus subtilis

  1. 1. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Jiangnan Ministry of Education; 
    2. School of Biotechnology, Jiangnan University, Wuxi 214122， China
  • Online:2016-08-18 Published:2016-08-18

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摘要： 内切β-N-乙酰氨基葡萄糖苷酶H(EndoH)是一类识别并切割与糖肽和糖蛋白的天冬氨酸残基连接的寡聚糖上N-乙酰氨基壳糖核心的糖苷酶，可用于糖组学中大规模糖基化位点的鉴定。克隆阿维链霉菌来源的endoH，成功构建pMA0911.1-EndoH重组质粒，并转化到枯草芽孢杆菌WB600中。发酵液经疏水层析和离子交换层析等技术手段得到目的蛋白EndoH。通过重组EndoH高效率酶切RNaseB及对鸡卵清标准糖蛋白(OVA)的N-糖链结构的鉴定，表明重组EndoH在糖组学N-糖链结构研究中具有良好的应用前景。

关键词: 内切&beta, -N-乙酰氨基葡萄糖苷酶H (EndoH)；RNaseB；鸡卵清标准糖蛋白(OVA)；N-糖链 

Abstract: Endo-β-N-acetylglucosaminidase H (Endo H) is a glycohydrolase which cleaves the β-1,4-glycosidic bonds of the N-acetylglucosamine core of oligosaccharides and leaves one N-acetylchitobiose attached to the asparagine residue of the glycoprotein. It can be used to identify the glycosylation sites. In this study, we cloned the endoH gene from the Streptomyces avermitilis, then subcloned into expression vector pMA0911.1. We expressed EndoH in Bacillus subtilis WB600. The fermentation broth was purified by hydrophobic chromatography and ionic exchange, and relatively purified EndoH was obtained. Utility of EndoH in digestion of RNaseB and identification of N-glycan structure in OVA indicated the potential of recombinant EndoH in research of N-glycan and glycomic.

Key words: endo-β-N-acetyglucosaminidase H(EndoH), RNaseB, the chicken ovalbumin standard glycoprotein (OVA), N-Glycan