[1]李 玲,凌 文,石牡丹,等.基于基因组的TEV蛋白酶的高表达[J].南京师大学报(自然科学版),2014,37(03):95.
　Li Ling,Ling Wen,Shi Mudan,et al.Chromosome-based TEV Protease Overexpression[J].Journal of Nanjing Normal University(Natural Science Edition),2014,37(03):95.
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基于基因组的TEV蛋白酶的高表达()
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《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

卷:
第37卷
期数:
2014年03期
页码:
95
栏目:
生命科学
出版日期:
2014-09-30

文章信息/Info

Title:
Chromosome-based TEV Protease Overexpression
作者:
李 玲; 凌 文; 石牡丹; 尚广东
南京师范大学生命科学学院,江苏省微生物工程技术研究中心,江苏省微生物与功能基因组学重点实验室,江苏 南京 210023
Author(s):
Li Ling; Ling Wen; Shi Mudan; Shang Guangdong
School of Life Sciences,Nanjing Normal University,Jiangsu Engineering and Technology Research Center for Microbiology,Jiangsu Key Laboratory for Microbes and Functional Genomics,Nanjing 210023,China
关键词:
基于基因组; 重组工程; TEV; 蛋白表达
Keywords:
chromosome-based; recombineering; TEV; protein expression
分类号:
Q819
文献标志码:
A
摘要:
TEV蛋白酶由于其高酶切活性、酶切位点的专一性和酶切条件的宽容性而在蛋白分离和蛋白质组学研究等领域有着广泛的应用.目前获得TEV蛋白酶的方法是将克隆在质粒上的TEV基因在大肠杆菌表达菌株BL21(DE3)中实现过表达.但本方法有一定缺点,如需使用抗生素,这就可能在后续的纯化中引入杂质,菌株群体中不均一性而降低产量等.本研究通过重组工程方法将受T7强启动子驱动的,与麦芽糖结合蛋白MBP融合的TEV蛋白酶基因整合至BL21(DE3)的基因组上进行过表达.MBP-TEV在细胞内自剪切获得分离的TEV.Ni-NTA亲和层析得到纯化的TEV,产量可达4.2 mg/L.所分离的TEV表达出良好的酶切活性.本菌株有着以之大规模获得TEV的潜力,所建立的通过重组工程将外源基因整合至BL21(DE3)基因组的进行表达的方法也可成为通用的平台技术.
Abstract:
Due to its high proteinase cleavage activity,specificity and effective in a wide range of conditions,Tobacco etch virus protease(TEV)has many applications,ranging from protein isolation to proteomics study.Currently,TEV is produced by plasmid-based overexpression in Escherichia coli BL21(DE3).Yet,the method has inherent disadvantages,for example,it needs antibiotic to maintain the plasmid which may bring impurities during the protein purification; and non-homogeneity of the strain population which may reduce the yield.Herein,we report the recombineering mediated integration of MBP fused TEV gene under the strong T7 promoter into E.coli BL21(DE3)chromosome.Intracellular digestion of chromosomal based overexpression of MBP-TEV released TEV which was subsequently isolated though Ni-NTA affinity purification,the yield of TEV was up to 4.2 mg/L.Purified TEV shows fine protease activity.The engineered strain has the potential to be used for large scale TEV purification,the established recombineering method can be a platform for the genome engineering and heterologus gene expression in E.coli BL21(DE3).

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备注/Memo

备注/Memo:
收稿日期:2013-11-09.
基金项目:国家自然科学基金(NSFC81273412).
通讯联系人:尚广东,副教授,研究方向:微生物药物的研发.E-mail:shanggd@hotmail.com
更新日期/Last Update: 2014-09-30