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N-糖酰胺酶F在大肠杆菌中的高效表达及其脱糖基化作用研究
引用本文:苏移山,王圣钧,王鹏,祁庆生.N-糖酰胺酶F在大肠杆菌中的高效表达及其脱糖基化作用研究[J].生物工程学报,2005,21(6):911-915.
作者姓名:苏移山  王圣钧  王鹏  祁庆生
作者单位:山东大学微生物技术国家重点实验室,济南,250100
基金项目:国家自然科学基金资助项目(No.30470399).
摘    要:从脑膜炎脓杆菌(Flavobacterium meningosepticum)基因组中通过PCR扩增了N-糖酰胺酶F(PNGase F)基因,经酶切后与表达载体pET28a连接,获得的重组质粒转入大肠杆菌BL21(DE3)。重组大肠杆菌经诱导表达和纯化提取后,获取大量高纯度N-糖酰胺酶F,其纯度达90%以上。试验证明,经纯化的重组N-糖酰胺酶F可以切除核糖核酸酶B、转铁蛋白和人IgG等糖蛋白上的N-糖链,具有脱糖基化作用。

关 键 词:N-糖酰胺酶F,  PET28a,  Rnase  B,  脱糖基化
文章编号:1000-3061(2005)06-0911-05
收稿时间:06 16 2005 12:00AM
修稿时间:08 31 2005 12:00AM

High Level Expression of PNGase F in Escherichia coli and its Bioactivities
SU Yi-Shan,WANG Sheng-Jun,WANG Peng,QI Qing-Sheng.High Level Expression of PNGase F in Escherichia coli and its Bioactivities[J].Chinese Journal of Biotechnology,2005,21(6):911-915.
Authors:SU Yi-Shan  WANG Sheng-Jun  WANG Peng  QI Qing-Sheng
Institution:Life Science School, Shandong University, Jinan 250100, China
Abstract:In order to obtain active recombinant PNGase F in Escherichia coli, a prokaryotic expression vector pET28a/PNGase F was constructed. Amplification of PNGase F was obtained using PCR technique employing suitable primers designed according to the PNGase F gene sequence from Flavobacterium meningosepticum. The expression of PNGase F gene in LB medium or M9 medium led to the formation of inclusion body and soluble protein, respectively. The refolding of the denatured inclusion body was successful by gradual dilution. Further purification of the refolded protein and soluble crude extract from M9 medium with Ni~ 2+-NTA argarose resulted a 90% purified PNGase F. The purified protein catalyzed the complete and intact cleavage of N-linked oligosaccharides from various glycoproteins. The efficiency of this cleavage was affected by the substrate status in the reaction system. In summary, we have developed an enzyme production system where PNGase F was over-expressed in recombinant E. coli. This system can provide more than 15mg/L purified active PNGase F. This purified active PNGase F can be used as tools in analyzing the oligosaccharide structure.
Keywords:PNGase F  pET28a  RNase B  deglycosylation
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