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Construction of Escherichia Coli Fusion Expression Vectors(PDF)

《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

Issue:
2013年03期
Page:
97-102
Research Field:
生命科学
Publishing date:

Info

Title:
Construction of Escherichia Coli Fusion Expression Vectors
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
Keywords:
fusion expression vectors; fusion tag; chaperone; TEV; N-acetyl-D-glucosamine 2-epimerase
PACS:
Q819
DOI:
-
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.

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Last Update: 2013-09-30