|Table of Contents|

Construction of Escherichia Coli Fusion Expression Vectors(PDF)


Research Field:
Publishing date:


Construction of Escherichia Coli Fusion Expression Vectors
Zhang FeifeiWang RuiqingZhu YupengMa ChaoShang 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
fusion expression vectorsfusion tagchaperoneTEVN-acetyl-D-glucosamine 2-epimerase
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.


[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.


Last Update: 2013-09-30