Gateway vectors for the production of combinatorially‐tagged His6‐MBP fusion proteins in the cytoplasm and periplasm of Escherichia coli

Many proteins that accumulate in the form of insoluble aggregates when they are overproduced in Escherichia coli can be rendered soluble by fusing them to E. coli maltose binding protein (MBP), and this will often enable them to fold in to their biologically active conformations. Yet, although it is an excellent solubility enhancer, MBP is not a particularly good affinity tag for protein purification. To compensate for this shortcoming, we have engineered and successfully tested Gateway destination vectors for the production of dual His6MBP‐tagged fusion proteins in the cytoplasm and periplasm of E. coli. The MBP moiety improves the yield and solubility of its fusion partners while the hexahistidine tag (His‐tag) serves to facilitate their purification. The availability of a vector that targets His6MBP fusion proteins to the periplasm expands the utility of this dual tagging approach to include proteins that contain disulfide bonds or are toxic in the bacterial cytoplasm.

[1]  Kazuhiro Yoshikawa,et al.  Production and characterization of an active single-chain variable fragment antibody recognizing CD25. , 2005, Cancer letters.

[2]  D. Waugh,et al.  Making the most of affinity tags. , 2005, Trends in biotechnology.

[3]  T. Copeland,et al.  Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro. , 2004, Protein expression and purification.

[4]  Naomi E Chayen,et al.  Turning protein crystallisation from an art into a science. , 2004, Current opinion in structural biology.

[5]  L. Goh,et al.  Soluble expression of a functionally active Plasmodium falciparum falcipain-2 fused to maltose-binding protein in Escherichia coli. , 2003, Protein expression and purification.

[6]  A. Chaffotte,et al.  Assistance of maltose binding protein to the in vivo folding of the disulfide-rich C-terminal fragment from Plasmodium falciparum merozoite surface protein 1 expressed in Escherichia coli. , 2003, Biochemistry.

[7]  K. M. Routzahn,et al.  Maltodextrin‐binding proteins from diverse bacteria and archaea are potent solubility enhancers , 2003, FEBS letters.

[8]  Yan-Ping Shih,et al.  High‐throughput screening of soluble recombinant proteins , 2002, Protein science : a publication of the Protein Society.

[9]  I. Huys,et al.  Purification, characterization and biosynthesis of parabutoxin 3, a component of Parabuthus transvaalicus venom. , 2002, European journal of biochemistry.

[10]  Martin Hammarström,et al.  Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli , 2002, Protein science : a publication of the Protein Society.

[11]  D. E. Anderson,et al.  Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. , 2001, Protein engineering.

[12]  C. Giuliani,et al.  Expression of an active recombinant lysine 49 phospholipase A(2) myotoxin as a fusion protein in bacteria. , 2001, Toxicon : official journal of the International Society on Toxinology.

[13]  Y. Berdichevsky,et al.  Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies. , 2001, Journal of molecular biology.

[14]  J. Lefèvre,et al.  A strategy for optimizing the monodispersity of fusion proteins: application to purification of recombinant HPV E6 oncoprotein. , 2001, Protein engineering.

[15]  R C Stevens,et al.  Design of high-throughput methods of protein production for structural biology. , 2000, Structure.

[16]  P. Riggs Expression and purification of recombinant proteins by fusion to maltose-binding protein , 2000, Molecular biotechnology.

[17]  B. Leiting,et al.  High-level expression of soluble protein in Escherichia coli using a His6-tag and maltose-binding-protein double-affinity fusion system. , 1997, Protein expression and purification.

[18]  W. Rutter,et al.  Engineering ribonuclease A: production, purification and characterization of wild-type enzyme and mutants at Gln11. , 1995, Protein engineering.

[19]  J. Brosius,et al.  Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. , 1983, Gene.

[20]  H. Neu,et al.  Release of Surface Enzymes in Enterobacteriaceae by Osmotic Shock , 1967, Journal of bacteriology.

[21]  J. Tropea,et al.  A generic method for the production of recombinant proteins in Escherichia coli using a dual hexahistidine-maltose-binding protein affinity tag. , 2007, Methods in molecular biology.

[22]  K. M. Routzahn,et al.  Differential effects of supplementary affinity tags on the solubility of MBP fusion proteins , 2004, Journal of Structural and Functional Genomics.

[23]  D. Waugh,et al.  Maltose-binding protein as a solubility enhancer. , 2003, Methods in molecular biology.

[24]  R. Raines,et al.  Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A. , 2000, European journal of biochemistry.

[25]  D. Waugh,et al.  Escherichia coli maltose‐binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused , 1999, Protein science : a publication of the Protein Society.

[26]  J. Tropea,et al.  Electronic Reprint Biological Crystallography Three-dimensional Structure of the Type Iii Secretion Chaperone Syce from Yersinia Pestis Biological Crystallography Three-dimensional Structure of the Type Iii Secretion Chaperone Syce from Yersinia Pestis , 2022 .