CAJ | 학술논문

目的：通过巴斯德毕赤酵母（ Pichia pastoris）表达制备内切β-N-乙酰氨基葡糖苷酶-H（ endo-beta-N-acetyl-glucosaminidase H ，Endo-H），并用于蛋白质的N-糖基化分析。方法根据GenBank中公布的褶皱链霉菌（ Strepto-myces plicatus） Endo-H的cDNA序列，设计并合成Endo-H全基因，将其克隆至P．pastoris表达载体pPIC9中，重组质粒转化P．pastoris JC308宿主菌，工程菌经甲醇诱导，分泌表达Endo-H，目的蛋白经疏水层析、凝胶过滤两步纯化后，纯度可＞95％。成品用于基于DNA测序的荧光辅助糖电泳（ DNA sequencer assisted fluorophore-assisted carbohy-drate electrophoresis，DSA-FACE）分析核糖核酸酶B（ribonuclease B，RNaseB）的糖基结构，比较了Endo-H与商品化N-糖酰胺酶F（peptide-N-asparigine amidase F，PNGase F）的糖基切割功能。结果利用P．pastoris表达制备Endo-H，可切割天然或变性状态下的β-1，4-糖苷键连接的甘露糖型结构糖链，不能切割复杂型糖链糖蛋白；经DSA-FACE分析，结果显示Endo-H酶切RNaseB后其糖链为Man5 GlcNAc-Man9 GlcNAc，PNGaseF酶切RNaseB的糖链为Man5 GlcNAc2-Man9 GlcNAc2，两者相差一个GlcNAc。结论通过P．pastoris制备的Endo-H具有天然生物活性，可用于蛋白质的N-糖基化结构分析。
목적：통과파사덕필적효모（ Pichia pastoris）표체제비내절β-N-을선안기포당감매-H（ endo-beta-N-acetyl-glucosaminidase H ，Endo-H），병용우단백질적N-당기화분석。방법근거GenBank중공포적습추련매균（ Strepto-myces plicatus） Endo-H적cDNA서렬，설계병합성Endo-H전기인，장기극륭지P．pastoris표체재체pPIC9중，중조질립전화P．pastoris JC308숙주균，공정균경갑순유도，분비표체Endo-H，목적단백경소수층석、응효과려량보순화후，순도가＞95％。성품용우기우DNA측서적형광보조당전영（ DNA sequencer assisted fluorophore-assisted carbohy-drate electrophoresis，DSA-FACE）분석핵당핵산매B（ribonuclease B，RNaseB）적당기결구，비교료Endo-H여상품화N-당선알매F（peptide-N-asparigine amidase F，PNGase F）적당기절할공능。결과이용P．pastoris표체제비Endo-H，가절할천연혹변성상태하적β-1，4-당감건련접적감로당형결구당련，불능절할복잡형당련당단백；경DSA-FACE분석，결과현시Endo-H매절RNaseB후기당련위Man5 GlcNAc-Man9 GlcNAc，PNGaseF매절RNaseB적당련위Man5 GlcNAc2-Man9 GlcNAc2，량자상차일개GlcNAc。결론통과P．pastoris제비적Endo-H구유천연생물활성，가용우단백질적N-당기화결구분석。
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.