[1]张飞飞,王瑞青,朱宇鹏,等.大肠杆菌系列融合表达载体的构建[J].南京师大学报(自然科学版),2013,36(03):97-102.
　Zhang Feifei,Wang Ruiqing,Zhu Yupeng,et al.Construction of Escherichia Coli Fusion Expression Vectors[J].Journal of Nanjing Normal University(Natural Science Edition),2013,36(03):97-102.
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大肠杆菌系列融合表达载体的构建()
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《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

卷:
第36卷
期数:
2013年03期
页码:
97-102
栏目:
生命科学
出版日期:
2013-09-30

文章信息/Info

Title:
Construction of Escherichia Coli Fusion Expression Vectors
作者:
张飞飞; 王瑞青; 朱宇鹏; 马 超; 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):
Zhang Feifei; Wang Ruiqing; Zhu Yupeng; Ma Chao; Shang Guangdong
Jiangsu Engineering and Technology Research Center for Microbiology,Jiangsu Key Laboratory for Microbes and Functional Genomics,School of Life Sciences,Nanjing Normal University,Nanjing 210023,China
关键词:
融合表达载体; 融合标签; 伴侣蛋白; TEV; N-乙酰-D-葡萄糖胺2-异构酶
Keywords:
fusion expression vectors; fusion tag; chaperone; TEV; N-acetyl-D-glucosamine 2-epimerase
分类号:
Q819
摘要:
许多异源蛋白在大肠杆菌内的表达是以不可溶、无生物学活性的包涵体形式存在,这为蛋白质的功能研究带来困难.融合表达是提高蛋白可溶性的有效方案之一.为构建通用型融合表达载体,本研究将5种常见的融合标签即突变型麦芽糖结合蛋白(mMBP)、小分子泛素样修饰蛋白(SUMO)、翻译起始因子(IF2-I)、氮源利用物质A(NusA)和谷胱甘肽转移酶(GST)以及3种伴侣蛋白(GroEL、DnaK和TF)分别克隆至pET30a(+),构建了系列融合表达载体.这些载体含有相同的克隆位点以及位于融合标签羧基端的烟草蚀刻病毒蛋白酶(TEV)的酶切位点.N-乙酰-D-葡萄糖胺2-异构酶基因hRnBP克隆到mMBP融合表达载体后,经IPTG诱导,SDS-PAGE检测表明融合蛋白均得到了高效表达且几乎完全可溶,TEV酶切获得了预期的带型.全细胞偶联生成N-乙酰-D-神经氨酸实验发现mMBP-hRnBP的摩尔转化率较无mMBP标签的体系提高了近60%,证明了融合表达载体中融合标签的适用性.新型的原核融合表达载体为蛋白的融合表达、分离纯化及功能研究提供了更多的选择.
Abstract:
Many heterologous proteins exhibit as insoluble,biologically inactive inclusion body form when overexpressed in Escherichia coli,thus hampering the functional study.Fusion expression is a practical means to achieve soluble expression.To obtain general fusion vectors,five fusion tags,including mutated maltose binding protein(mMBP),small ubiquitin-related modifier(SUMO),initiation factor 2-I(IF2-I),N utilization substance A(NusA)and glutathione S-transferase(GST); and three chaperones,including GroEL,DanK and TF were each cloned into pET30a(+),obtaining serial fusion expression vectors.These expression vectors share the same cloning sites with tobacco etch virus protease(TEV)digestion site engineered after carboxyl terminus of fusion tag.N-acetyl-D-glucosamine 2-epimerase gene hRNBP was cloned into mMBP fusion vector,after IPTG induction,SDS-PAGE analysis showed that high-level expression and nearly all soluble proteins were obtained.Expected digestion patterns were achieved after TEV digestions.Comparison between the coupled well cell biocatalysis of N-acetyl-D-neuraminic acid shows that the molar conversion rate of mMBP-hRnBP is nearly 60% higher than that of hRnBP,demonstrating the newly developed expression vectors have potential for the broad applications in protein expression,isolation and function investigation.

参考文献/References:

[1] Waugh D S.Making the most of affinity tags[J].Trends Biotechnol,2005,23(6):316-320.
[2]Houry W A.Chaperone-assisted protein folding in the cell cytoplasm[J].Curr Protein Pept Sci,2001,2(3):227-244.
[3]Guana C,Lib P,Rigssa P D,et al.Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein[J].Gene,1988,67(1):21-30.
[4]Marblestone J G,Edavettal S C,Lim Y,et al.Comparison of SUMO fusion technology with traditional gene fusion systems:Enhanced expression and solubility with SUMO[J].Protein Sci,2006,15(1):182-189.
[5]Sorensen H P,Sperling-Petersen H U,Mortensen K K.A favorable solubility partner for the recombinant expression of streptavidin[J].Protein Expr Purif,2003,32(2):252-259.
[6]De Marco V,Stier G,Blandin S,et al.The solubility and stability of recombinant proteins are increased by their fusion to NusA[J].Biochem Biophys Res Commun,2004,322(3):766-771.
[7]Wu X,Oppermann U.High-level expression and rapid purification of rare-codon genes from hyperthermophilic archaea by the GST gene fusion system[J].J Chromatogr B,2003,786(1):177-185.
[8]Walker I H,Hsieh P,Riggs P D.Mutations in maltose-binding protein that alter affinity and solubility properties[J].Appl Microbiol Biotechnol,2010,88(1):187-197.
[9]Kyratsous C A,Silverstein S J,DeLong C R,et al.Chaperone-fusion expression plasmid vectors for improved solubility of recombinant proteins in Escherichia coli[J].Gene,2009,440(1/2):9-15.
[10]Deuerling E,Schulze-Specking A,Tomoyasu T,et al.Trigger factor and DnaK cooperate in folding of newly synthesized proteins[J].Nature,1999,400(6745):693-696.
[11]Thapa A,Shahnawaz M,Karki P,et al.Purification of inclusion body-forming peptides and proteins in soluble form by fusion to Escherichia coli thermostable proteins[J].Biotechniques,2008,44(6):787-796.
[12]Nishihara K,Kanemori M,Yanagi H,et al.Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli[J].Appl Environ Microbiol,2000,66(3):884-889.
[13]Kapust R B,Tozser J,Fox J D,et al.Tobacco etch virus protease:mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency[J].Protein Eng,2001,14(12):993-1 000.
[14]Sambrook J,Fritsch E F,Maniatis T.Molecular Cloning:A Laboratory Manual[M].2nd ed.New York:Cold Spring Harbor Laboratory Press,1989:16-34.

备注/Memo

备注/Memo:
收稿日期:2012-12-10.
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: 2013-09-30