Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research
  • Published:

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 volume 13, pages 366–372 (1995)Cite this article

Abstract

We have developed a system for probing protein/protein interactions which makes use of the bacterial flagellum to display random peptide libraries on the surface of E. coli. In developing the system the entire coding sequence of E. coli thioredoxin (trxA) was inserted into a dispensable region of the gene for flagellin (fliC), the major structural component of the E. coli flagellum. The resulting fusion protein (FLITRX) was efficiently exported and assembled into partially functional flagella on the bacterial cell surface. A diverse library of random dodecapeptides were displayed in FLITRX on the exterior of E. coli as conformationally constrained insertions into the thioredoxin active-site loop, a location known to be a highly permissive site for the insertion of exogenous peptide sequences into native thioredoxin. To demonstrate that members of this library could be bound and selected via specific protein/protein interactions to a target protein, a method was devised to enable efficient isolation of those bacteria displaying peptides with affinity to immobilized antibodies. We have unambiguously mapped three different antibody epitopes using this method. Peptides selected as FLITRX active-site fusions retain their binding specificity when made as native thioredoxin active-site loop fusions. This will facilitate future structural characterizations and broaden the general utility of the system for exploring other classes of protein-protein interactions.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Smith, G.P. 1985. Filamentaous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228: 1315–1317.

    Article  CAS  Google Scholar 

  2. Parmley, S.F. and Smith, G.P. 1988. Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene 73: 305–318.

    Article  CAS  Google Scholar 

  3. Scott, J.K. and Craig, L. 1994. Random peptide libraries. Curr. Opin. Biotechnol. 5: 40–48.

    Article  CAS  Google Scholar 

  4. Hammer, J., Takacs, B. and Sinigaglia, F. 1992. Identification of a motif for HLA-DR1 binding peptides using M13 display libraries. J. Exp. Med. 176: 1007–1013.

    Article  CAS  Google Scholar 

  5. Oldenberg, K.R., Loganathan, D., Goldstein, I.J., Schultz, P.G. and Gallop, M.A. 1992. Peptide ligands for a sugar-binding protein isolated from a random peptide library. Proc. Nat. Acad. Sci. USA 89: 5393–5397.

    Article  Google Scholar 

  6. Scott, J.K., Loganathan, D., Easley, R.B., Gong, X. and Goldstein, I.J. 1992. A family of concanavalin A-binding peptides from a hexapeptide epitope library. Proc. Nat. Acad. Sci. USA 89: 5398–5402.

    Article  CAS  Google Scholar 

  7. Blond-Elguindi, S., Cwirla, S.E., Dower, W.J., Lipshutz, R.J., Sprang, S.R., Sambrook, J.F. and Gething, M.-J.H. 1993. Affinity panning a library of peptides displayed on bacteriophage reveals the binding specificity of BiP. Cell 75: 717–728.

    Article  CAS  Google Scholar 

  8. Djojonegoro, B.M., Benedik, M.J. and Willson, R.C. 1994. Bacteriophage surface display of an immunoglobulin-binding domain of Staphylococcus aureus protein A. Bio/Technology 12: 169–172.

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. 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 circumvents inclusion body formation in the E. coli cytoplasm. Bio/Technology 11: 1187–193.

    Google Scholar 

  11. Scott, J.K. and Smith, G.P. 1990. Searching for peptide ligands with an epitope library. Science 249: 386–390.

    Article  CAS  Google Scholar 

  12. LaVallie, E.R. and Stahl, M.L. 1989. Cloning of the flagellin gene from Bacillus subtilis and complementation studies of an in vitro-derived deletion mutation. J. Bacteriol. 171: 3085–3094.

    Article  CAS  Google Scholar 

  13. Namba, K., Yamashita, I. and Vonderviszt, F. 1989. Structure of the core and central channel of bacterial flagella. Nature 342: 648–654.

    Article  CAS  Google Scholar 

  14. Wilson, A.R. and Beveridge, T.J. 1993. Bacterial fiagellar filaments and their component flagellins. Can. J. Microbiol. 39: 451–472.

    Article  CAS  Google Scholar 

  15. Kuwajima, G. 1988. Construction of a minimum-size functional flagellin of Escherichia coli. J. Bacteriol. 170: 3305–3309

    Article  CAS  Google Scholar 

  16. Joys, T.M. 1988. The flagellar filament protein. Can. J. Microbiol. 34: 452–458.

    Article  CAS  Google Scholar 

  17. Diderichsen, B. 1980. flu, a metastable gene controlling surface properties of Escherchia coli. J. Bacteriol. 141: 858–867.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Ofek, I. and Beachy, E.H. 1978. Mannose binding and epithelial cell adherence of Escherichia coli. Infect. Immun. 22: 247–254.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Ponniah, S., Endres, R.O., Hasty, D.L. and Abraham, S.N. 1991. Fragmentation of E. coli type 1 fimbriae exposes cryptic D-mannose-binding sites. J. Bacteriol. 173: 4195–4202.

    Article  CAS  Google Scholar 

  20. Clore, G.M., Apella, E., Yamada, M., Matsushima, K. and Gronenborn, A.M. 1990. Three dimensional structure of interleukin 8 in solution. Biochemistry 29: 1689–1696.

    Article  CAS  Google Scholar 

  21. Hutchcroft, J.E., Anostario, M.,Jr., Harrison, M.L. and Geahlen, R.L. 1991. Renaturation and assay of protein kinases after electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Method in Enzymol. 200: 417–423.

    Article  CAS  Google Scholar 

  22. Shackelford, D.A. and Zivin, J.A. 1993. Renaturation of calcium/calmodulin-dependent protein kinase activity after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels to membranes. Anal. Biochem. 211: 131–138.

    Article  CAS  Google Scholar 

  23. Charbit, A., Molla, A., Saurin, W. and Hofnung, M. 1988. Versatility of a vector for expressing foreign polypeptides at the surface of Gram-negative Bacteria. Gene 70: 181–189.

    Article  CAS  Google Scholar 

  24. Brown, S. 1992. Engineered iron oxide-adhesion mutants of the Escherichia coli phage λ receptor. Proc. Natl. Acad. Sci. USA 89: 8651–8655.

    Article  CAS  Google Scholar 

  25. Francisco, J.A., Campbell, R., Iverson, B.L. and Georgiou, G. 1993. Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface. Proc. Natl. Acad. Sci. USA 90: 10444–10448.

    Article  CAS  Google Scholar 

  26. McLafferty, M.A., Kent, R.B., Ladner, R.C. and Markland, W. 1993. M13 bacteriophage displaying disulfide-constrained microproteins. Gene 128: 29–36.

    Article  CAS  Google Scholar 

  27. Fedorov, O.V. and Efimov, A.V. 1990. Flagellin as an object for supramolecular engineering. Protein Eng. 3: 411–413.

    Article  CAS  Google Scholar 

  28. Jessen, T., Cohen, B., Colas, P., Mendelsohn, A.M., McCoy, J. and Brent, R. 1994. Artificial peptide inhibitors of Cdk2-dependent cell proliferation. Manuscript in preparation.

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

    Article  CAS  Google Scholar 

  30. Wurfel, M., Haberlein, I and Follmann, H. 1993. Facile sulfitolysis of the disulfide bonds in oxidized thioredoxin and glutaredoxin. Eur. J. Biochem. 211: 609–614.

    Article  CAS  Google Scholar 

  31. Brent, R. and Ptashne, M. 1981. Mechanism of action of the lex A gene product. Proc. Nat. Acad. Sci. USA 78: 4204–4208.

    Article  CAS  Google Scholar 

  32. Blair, D.F., Kim, D.Y. and Berg, H.C. 1991. Mutant MotB proteins in Escherichia coli Bacteriol. 173: 4049–4055.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Author information

Author notes

  1. John M. McCoy: Corresponding author.

Authors and Affiliations

  1. Genetics Institute, Inc., 87 Cambridgepark Drive, Cambridge, MA, 02140

    Zhijian Lu, Kristin S. Murray, Victor Van Cleave, Edward R. LaVallie, Mark L. Stahl & John M. McCoy

Authors
  1. Zhijian Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kristin S. Murray
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Victor Van Cleave
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Edward R. LaVallie
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mark L. Stahl
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. John M. McCoy
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lu, Z., Murray, K., Cleave, V. et al. 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. Nat Biotechnol 13, 366–372 (1995). https://doi.org/10.1038/nbt0495-366

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0495-366

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing