| 摘要: |
| N-糖酰胺酶F(PNGase F)是由革兰氏阴性菌——脑膜炎脓杆菌分泌的一种分子量为34.8 kDa的去糖基化酶.本研究通过PCR技术扩增出945 bp的N-糖酰胺酶F基因,经酶切、连接,构建了大肠杆菌表达载体pET28a-PNGase F.将 pET28a-PNGase F 转入大肠杆菌BL21中,16 ℃诱导表达及Ni柱纯化后,SDS-PAGE鉴定,获得超过95%纯度的可溶性表达的N-糖酰胺酶F.对Na+依赖的中性氨基酸转运蛋白1(SNAT1)和2(SNAT2)进行脱糖基化作用检测,结果证明:纯化的N-糖酰胺酶F对SNAT1和SNAT2具有高效的脱糖基化作用. |
| 关键词: N-糖酰胺酶 F 可溶性表达与纯化 中性氨基酸转运蛋白SNAT 脱糖基化 |
| DOI： |
| 分类号: |
| 基金项目:国家自然基金项目(31270883);上海市科学技术委员会项目(10540503400);上海市教育委员会科研创新项目(13ZZ103) |
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| Expression and purification of PNGase F( PNGase F) in escherichia coli and its functional study |
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MIN Weiyong, LIU Li, ZHANG Zhou
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College of Life and Environment Sciences,Shanghai Normal University
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| Abstract: |
| N-glycosidase F(PNGase F)is a enzyme of deglycosylation,secreted by the gram-negative bacterium Flavobacterium meningosepticum.It’s theoretical molecular weight is about 34.8 kDa.In this study,the PNGase F gene of 945 bp was amplified by PCR technique.A prokaryotic expression vector,pET28a-PNGase F,was constructed by digestion and ligation.After E.coli(BL21)containing pET28a-PNGase F plasmids was cultured and induced by IPTG at 16 ℃,soluble PNGase F was purified through Ni column,and the purity is up to over 95% by SDS-PAGE identification.Deglycosylation of purified PNGase F on neutral amino acid transporter SNAT1 and SNAT2 showed that PNGase F is highly effective and both SNAT1 and SNAT2 contain glycosylated sites. |
| Key words: N-glycosidase F soluable expression and purification neutal amino acid transporter SNAT deglycosylation |
