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.

Expression of high-affinity human antibody fragments in bacteria

Nature Protocols volume 7, pages 364–373 (2012)Cite this article

Subjects

Abstract

Here we describe protocols for the expression of human antibody fragments in Escherichia coli. Antigen-specific clones are identified by soluble fragment ELISA and concentrated by periplasmic preparation. They are then further purified by affinity chromatography. This article provides an overview of expression and purification strategies for human antibody fragments, as well as detailed protocols for the identification of high-affinity binders and for affinity maturation.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: Analysis of sample purity by SDS-PAGE.
Figure 2: Analysis of sample purity by size-exclusion chromatography.

References

  1. Choy, E.H. et al. Efficacy of a novel PEGylated humanized anti-TNF fragment (CDP870) in patients with rheumatoid arthritis: a phase II double-blinded, randomized, dose-escalating trial. Rheumatology (Oxford) 41, 1133–1137 (2002).

    Article  CAS  Google Scholar 

  2. Cabilly, S. et al. Generation of antibody activity from immunoglobulin polypeptide chains produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 81, 3273–3277 (1984).

    Article  CAS  Google Scholar 

  3. Skerra, A. & Pluckthun, A. Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science 240, 1038–1041 (1988).

    Article  CAS  Google Scholar 

  4. Wulfing, C. & Pluckthun, A. Protein folding in the periplasm of Escherichia coli. Mol. Microbiol. 12, 685–692 (1994).

    Article  CAS  Google Scholar 

  5. Power, B.E. et al. High-level temperature-induced synthesis of an antibody VH-domain in Escherichia coli using the PelB secretion signal. Gene 113, 95–99 (1992).

    Article  CAS  Google Scholar 

  6. Steiner, D., Forrer, P., Stumpp, M.T. & Pluckthun, A. Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. Nat. Biotechnol. 24, 823–831 (2006).

    Article  CAS  Google Scholar 

  7. Thie, H., Schirrmann, T., Paschke, M., Dubel, S. & Hust, M. SRP and Sec pathway leader peptides for antibody phage display and antibody fragment production in E. coli. Nat. Biotechnol. 25, 49–54 (2008).

    CAS  Google Scholar 

  8. Buchner, J., Brinkmann, U. & Pastan, I. Renaturation of a single-chain immunotoxin facilitated by chaperones and protein disulfide isomerase. Biotechnology (NY) 10, 682–685 (1992).

    CAS  Google Scholar 

  9. Lilie, H., Lang, K., Rudolph, R. & Buchner, J. Prolyl isomerases catalyze antibody folding in vitro. Protein Sci. 2, 1490–1496 (1993).

    Article  CAS  Google Scholar 

  10. Lowe, D. et al. Aggregation, stability, and formulation of human antibody therapeutics. Adv. Protein Chem. Struct. Biol. 84, 41–61 (2011).

    Article  CAS  Google Scholar 

  11. Rothlisberger, D., Honegger, A. & Pluckthun, A. Domain interactions in the Fab fragment: a comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability. J. Mol. Biol. 347, 773–789 (2005).

    Article  Google Scholar 

  12. Baneyx, F. Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 10, 411–421 (1999).

    Article  CAS  Google Scholar 

  13. Lo, B.K.C. Antibody Engineering: Methods and Protocols (Humana Press, 2004).

  14. Jansson, B., Uhlen, M. & Nygren, P.A. All individual domains of staphylococcal protein A show Fab binding. FEMS Immunol. Med. Microbiol. 20, 69–78 (1998).

    Article  CAS  Google Scholar 

  15. Bjorck, L. Protein L. A novel bacterial cell wall protein with affinity for Ig L chains. J. Immunol. 140, 1194–1197 (1988).

    CAS  PubMed  Google Scholar 

  16. Jonsson, U. et al. Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. Biotechniques 11, 620–627 (1991).

    CAS  PubMed  Google Scholar 

  17. Stenberg, E., Persson, B., Roos, H. & Urbaniczky, C. Quantitative-determination of surface concentration of protein with surface-plasmon resonance using radiolabeled proteins. J. Colloid Interf. Sci. 143, 513–526 (1991).

    Article  CAS  Google Scholar 

  18. Peters, W.B., Frasca, V. & Brown, R.K. Recent developments in isothermal titration calorimetry label free screening. Comb. Chem. High Throughput Screen 12, 772–790 (2009).

    Article  CAS  Google Scholar 

  19. Godber, B. et al. Direct quantification of analyte concentration by resonant acoustic profiling. Clin. Chem. 51, 1962–1972 (2005).

    Article  CAS  Google Scholar 

  20. Schnerr, H.R. Lead identification and optimization in crude samples using label free resonant acoustic profiling. J. Mol. Recognit. 23, 597–603 (2010).

    Article  CAS  Google Scholar 

  21. Abdiche, Y., Malashock, D., Pinkerton, A. & Pons, J. Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet. Anal. Biochem. 377, 209–217 (2008).

    Article  CAS  Google Scholar 

  22. Hawkins, R.E., Russell, S.J. & Winter, G. Selection of phage antibodies by binding affinity. Mimicking affinity maturation. J. Mol. Biol. 226, 889–896 (1992).

    Article  CAS  Google Scholar 

  23. Lee, C.M., Iorno, N., Sierro, F. & Christ, D. Selection of human antibody fragments by phage display. Nat. Protoc. 2, 3001–3008 (2007).

    Article  CAS  Google Scholar 

  24. Sambrook, J. & Russell, D.W. The Condensed Protocols from Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2006).

  25. Jager, M., Gehrig, P. & Pluckthun, A. The scFv fragment of the antibody hu4D5-8: evidence for early premature domain interaction in refolding. J. Mol. Biol. 305, 1111–1129 (2001).

    Article  CAS  Google Scholar 

  26. Christ, D., Famm, K. & Winter, G. Repertoires of aggregation-resistant human antibody domains. Protein Eng. Des. Sel. 20, 413–416 (2007).

    Article  CAS  Google Scholar 

  27. Dudgeon, K., Famm, K. & Christ, D. Sequence determinants of protein aggregation in human VH domains. Protein Eng. Des. Sel. 22, 217–220 (2009).

    Article  CAS  Google Scholar 

  28. Famm, K., Hansen, L., Christ, D. & Winter, G. Thermodynamically stable aggregation-resistant antibody domains through directed evolution. J. Mol. Biol. 376, 926–931 (2008).

    Article  CAS  Google Scholar 

  29. Miroux, B. & Walker, J.E. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Protocols are based on methods originally developed by D. Christ and others in G. Winter's group at the UK Medical Resarch Council (MRC) Laboratory of Molecular Biology. These were further modified at the Garvan Institute of Medical Research and at MedImmune. This work was funded by the Garvan Institute of Medical Research; MedImmune; the Australian National Health and Medical Council; the Australian Research Council; the Cancer Institute, NSW; and the UK MRC.

Author information

Authors and Affiliations

  1. Garvan Institute of Medical Research, Darlinghurst, Sydney, New South Wales, Australia

    Romain Rouet, Kip Dudgeon, Brendan Roome, Peter Schofield, David Langley, Peter Whitfeld & Daniel Christ

  2. The University of New South Wales, Faculty of Medicine, St. Vincent's Clinical School, Darlinghurst, Sydney, New South Wales, Australia

    Romain Rouet, Kip Dudgeon, Brendan Roome & Daniel Christ

  3. MedImmune, Cambridge, UK

    David Lowe, John Andrews & Lutz Jermutus

Authors
  1. Romain Rouet
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David Lowe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kip Dudgeon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Brendan Roome
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter Schofield
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. David Langley
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. John Andrews
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Peter Whitfeld
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Lutz Jermutus
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Daniel Christ
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D. Lowe, K.D., B.R., P.S., D. Langley, J.A. and P.W. wrote the paper. R.R. wrote the paper and generated figures. L.J. and D.C. wrote the paper and supervised research.

Corresponding author

Correspondence to Daniel Christ.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Rouet, R., Lowe, D., Dudgeon, K. et al. Expression of high-affinity human antibody fragments in bacteria. Nat Protoc 7, 364–373 (2012). https://doi.org/10.1038/nprot.2011.448

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.448

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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