军事医学Issue(3)：193-197,5.DOI:10.7644/j.issn.1674-9960.2014.03.008
Endo-H在毕赤酵母中的表达、纯化及其在N-糖基化分析中的应用
Expression of endo-beta-N-acetylglucosaminidase H in Pichia pastoris and its applica-tion to N-glycosylation analysis
摘要
Abstract
Objective To prepare endo-beta-N-acetylglucosaminidase H (Endo-H) expressed in Pichia pastoris, and apply it to N-glycosylation analysis .Methods One complete gene was synthesized on the basis of the cDNA sequence encoding Streptomyces plicatus reported in GenBank .The gene was cloned into the expression vector pPIC 9.The expression vector pPIC9-Endo-H was transformed into P.pastoris(JC308).The expression products were induced by methanol , puri-fied by two-step chromatography , used to analyze the glycan structures of RNaseB by the DNA sequencer assisted fluoro-phore-assisted carbohydrate electrophoresis (DSA-FACE)methods, and finally compared with peptide-N-asparagine amidase F(PNGase F).Results This enzyme expressed in P.pastoris(JC308) had the ability to hydrolyze natural or denatured high-mannose type of oligosaccharide linked by β-1,4-glycosidic bonds , but not complex-type oligosaccharide .The result of DSA-FACE showed that carbohydrate chains of Man 5 GlcNAc-Man9 GlcNAc could be obtained when RNaseB was hydrolyzed by Endo-H, and that Man5 GlcNAc2-Man9 GlcNAc2 chains became available when RNaseB was hydrolyzed by PNGase F . Conclusion Endo-H expressed in P.pastoris has bioactivity which can be used to analyze N-glycosylation with the method of DSA-FACE.关键词
内切β-N-乙酰氨基葡糖苷酶H/毕赤酵母/N-糖基化Key words
endo-beta-N-acetylglucosaminidase H/Pichia/N-glycosylation分类
医药卫生引用本文复制引用
支艳艳,唱韶红,巩新,宋西勇,吴军,刘波..Endo-H在毕赤酵母中的表达、纯化及其在N-糖基化分析中的应用[J].军事医学,2014,(3):193-197,5.基金项目
国家自然科学基金青年科学基金资助项目 （）
