Skip to main content
SpringerLink
Log in

Thioredoxin and Related Proteins as Multifunctional Fusion Tags for Soluble Expression in E. coli

  • Protocol
E. coliGene Expression Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 205))

Abstract

Escherichia coli has traditionally been a popular host for the production of heterologous proteins because of its ease of genetic manipulation and growth. Recombinant proteins produced in E. coli have been useful for a variety of purposes, including the study of protein tertiary structure, structure-function experiments, enzymology, and as bio-pharmaceuticals. Despite an impressive body of literature describing the production of numerous non-native proteins in E. coli, successful results are in no way assured. The use of E. coli as a robust expression system has been hampered by several integral pitfalls. Low or undetectable expression levels can often be caused by inefficient translation initiation of eukaryotic mRNAs on bacterial ribosomes (1). Recombinant proteins produced in E. coli sometimes retain the N-terminal initiator methionine residue, as they may be a poor substrate for the host methionine aminopeptidase (2). In addition, individual purification schemes must be devised for each native recombinant protein produced in E. coli. But perhaps most importantly, it is very common for recombinant proteins expressed in E. coli to form insoluble, misfolded cytoplasmic complexes known as “inclusion bodies” (3). The likelihood of inclusion body formation is unpredictable but appears to increase in proportion to the size and complexity of the protein.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Stormo, G. D., Schneider, T. D., and Gold, L. (1982) Characterization of translation initiation sites in E. coli. Nucleic Acids Res. 10, 2971–2996.

    Article  CAS  Google Scholar 

  2. Hirel, P. H., Schmitter, M. J., Dessen, P., Fayat, G., and Blanquet, S. (1989) Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc. Natl. Acad. Sci. USA 86, 8247–8251.

    Article  PubMed  CAS  Google Scholar 

  3. Mitraki, A. and King, J. (1989) Protein folding intermediates and inclusion body formation. Bio/Technology 7, 690–697.

    Article  CAS  Google Scholar 

  4. LaVallie, E. R., DiBlasio, E. A., Kovacic, S., Grant, K. L., Schendel, P. F., and McCoy, J. M. (1993) A thioredoxin gene fusion expression system that circum-vents inclusion body formation in the E. coli cytoplasm. Bio/Technology 11, 187–193.

    CAS  Google Scholar 

  5. Collins-Racie, L. A., McColgan, J. M., Grant, K. L., DiBlasio-Smith, E. A., McCoy, J. M., and LaVallie, E. R. (1995) Production of recombinant bovine enterokinase catalytic subunit in Escherichia coli using the novel secretory fusion partner DsbA. Bio/Technology 13, 982–987.

    Article  PubMed  CAS  Google Scholar 

  6. Lu, Z., DiBlasio-Smith, E. A., Grant, K. L., et al. (1996) “Histidine-patch” thioredoxins: Mutant forms of thioredoxin with metal chelating affinity that provide for convenient purifications of thioredoxin fusion proteins. J. Biol. Chem. 271, 5059–5065.

    Article  PubMed  CAS  Google Scholar 

  7. Smith, P. A., Tripp, B. C., DiBlasio-Smith, E. A., Lu, Z., LaVallie, E. R., and McCoy, J. M. (1998) A plasmid expression system for quantitative in vivo biotinylation of thioredoxin fusion proteins in Escherichia coli. Nucleic Acids Res. 26, 1414–1420.

    Article  CAS  Google Scholar 

  8. Lunn, C. A., Kathju, S., Wallace, B. J., Kushner, S. R., and Pigiet, V. (1984) Amplification and purification of plasmid-encoded thioredoxin from Escherichia coli K12. J. Biol. Chem. 259, 10,469–10,474.

    CAS  Google Scholar 

  9. Katti, S. K., LeMaster, D. M., and Eklund, H. (1990) Crystal structure of thioredoxin from Escherichia coli at 1.68 angstroms resolution. J. Mol. Biol. 212, 167–184.

    Article  PubMed  CAS  Google Scholar 

  10. Bardwell, J. C. A., McGovern, K., and Beckwith, J. (1991) Identification of a protein required for disulfide bond formation in vivo. Cell 67, 581–589.

    Article  PubMed  CAS  Google Scholar 

  11. Mazzarella, R. A., Srinivasan, M., Haugejorden, S. M., and Green, M. (1990) ERp72, an abundant luminal endoplasmic reticulum protein, contains three copies of the active site sequences of protein disulfide isomerase. J. Biol. Chem. 265, 1094–1101.

    PubMed  CAS  Google Scholar 

  12. LaVallie, E. R., Lu, Z., DiBlasio-Smith, E. A., Collins-Racie, L. A., and McCoy, J. M. (2000) Thioredoxin as a fusion partner for soluble recombinant protein production in Escherichia coli. Methods Enzymol. 326, 322–340.

    CAS  Google Scholar 

  13. Silen, J. L., Frank, D., Fujishige, A., Bone, R., and Agard, D. A. (1989) Analysis of prepro-α-lytic protease expression in Escherichia coli reveals that the pro region is required for activity. J. Bacteriol. 171, 1320–1325.

    PubMed  CAS  Google Scholar 

  14. Shinde, U., Chatterjee, S., and Inouye, M. (1993) Folding pathway mediated by an intramolecular chaperone. Proc. Natl. Acad. Sci. USA 90, 6924–6928.

    Article  PubMed  CAS  Google Scholar 

  15. Norrander, J., Kempe, T., and Messing, J. (1983) Construction of improved M13 vectors using oligonucleotide-directed mutagenesis. Gene 26, 101–106.

    Article  PubMed  CAS  Google Scholar 

  16. Shimatake, H. and Rosenberg, M. (1981) Purified λ regulatory protein cII positively activates promoters for lysogenic development. Nature 292, 128–132.

    Article  CAS  Google Scholar 

  17. Colas, P., Cohen, B., Jessen, T., Grishina, I., McCoy, J., and Brent, R. (1996) Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2. Nature 380, 548–550.

    Article  PubMed  CAS  Google Scholar 

  18. Lu, Z., Murray, K. S., Van Cleave, V., LaVallie, E. R., Stahl, M. L., and McCoy, J. M. (1995) Expression of thioredoxin random peptide libraries on the Escherichia coli cell surface as functional fusions to flagellin: a system designed for exploring protein-protein interactions. Bio/Technology 13, 366–372.

    Article  PubMed  CAS  Google Scholar 

  19. Tripp, B. C., Lu, Z., Bourque, K., Sookdeo, H., and McCoy, J. M. (2001) Investigation of the’ switch-epitope' concept with random peptide libraries displayed as thioredoxin loop fusions. Protein Eng. 14, 367–377.

    Article  PubMed  CAS  Google Scholar 

  20. Smith, D. B. and Johnson, K. S. (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 31–40.

    Article  PubMed  CAS  Google Scholar 

  21. di Guan, C., Li, P., Riggs, P. D., and Inouye, H. (1988) Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene 67, 21–30.

    Article  PubMed  Google Scholar 

  22. Porath, J. (1992) Immobilized metal ion affinity chromatography. Protein Expr. Purif. 3, 263–281.

    Article  PubMed  CAS  Google Scholar 

  23. Schatz, P. J. (1993) Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli. Bio/Technology 11, 1138–1143.

    CAS  Google Scholar 

  24. Hoffman, C. S. and Wright, A. (1985) Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion. Proc. Natl. Acad. Sci. USA 82, 5107–5111.

    Article  PubMed  CAS  Google Scholar 

  25. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  26. Brent, R. and Ptashne, M. (1981) Mechanism of action of the lexA gene product. Proc. Natl. Acad. Sci. USA 78, 4204–4208.

    Article  PubMed  CAS  Google Scholar 

  27. Mieschendahl, M., Petri, T., and Hanggi, U. (1986) A novel prophage independent trp regulated lambda pL expression system. Bio/Technology 4, 802–808.

    Article  CAS  Google Scholar 

  28. Lunn, C. A. and Pigiet, V. P. (1982) Localization of thioredoxin from Escherichia coli in an osmotically sensitive compartment. J. Biol. Chem. 257, 11,424–11, 430.

    Google Scholar 

  29. Holmgren, A. (1985) Thioredoxin. Ann. Rev. Biochem. 54, 237–271.

    Article  PubMed  CAS  Google Scholar 

  30. Maroux, S., Baratti, J., and Desnuelle, P. (1971) Purification and specificity of porcine enterokinase. J. Biol. Chem. 246, 5031–5039.

    PubMed  CAS  Google Scholar 

  31. LaVallie, E. R., Rehemtulla, A., Racie, L. A., et al. (1993) Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase. J. Biol. Chem. 268, 23,311–23,317.

    PubMed  CAS  Google Scholar 

  32. Dower, W. J., Miller, J. F., and Ragsdale, C. W. (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucl. Acids Res. 16, 6127–6145.

    Article  PubMed  CAS  Google Scholar 

  33. Schein, C. H. and Noteborn, M. H. M. (1988) Formation of soluble recombinant proteins in Escherichia coli is favored by lower growth temperature. Bio/Technology 6, 291–294.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Genetics Institute/Wyeth Research, Cambridge, MA

    Edward R. LaVallie, Elizabeth A. DiBlasio-Smith, Lisa A. Collins-Racie & Zhijian Lu

  2. Biogen, Inc., Cambridge, MA

    John M. McCoy

Authors
  1. Edward R. LaVallie
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elizabeth A. DiBlasio-Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lisa A. Collins-Racie
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhijian Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. John M. McCoy
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Applied Molecular Evolution, San Diego, CA, USA

    Peter E. Vaillancourt

Rights and permissions

Reprints and permissions

Copyright information

© 2003 Humana Press Inc.

About this protocol

Cite this protocol

LaVallie, E.R., DiBlasio-Smith, E.A., Collins-Racie, L.A., Lu, Z., McCoy, J.M. (2003). Thioredoxin and Related Proteins as Multifunctional Fusion Tags for Soluble Expression in E. coli . In: Vaillancourt, P.E. (eds) E. coliGene Expression Protocols. Methods in Molecular Biology™, vol 205. Humana Press. https://doi.org/10.1385/1-59259-301-1:119

Download citation

  • DOI: https://doi.org/10.1385/1-59259-301-1:119

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-008-3

  • Online ISBN: 978-1-59259-301-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation