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.

  • Article
  • Published:

Growth factor engineering by degenerate homoduplex gene family recombination

Nature Biotechnology volume 20, pages 1246–1250 (2002)Cite this article

Abstract

There is great interest in engineering human growth factors as potential therapeutic agonists and antagonists. We approached this goal with a synthetic DNA recombination method. We aligned a pool of “top-strand” oligonucleotides incorporating polymorphisms from mammalian genes encoding epidermal growth factor (EGF) using multiple polymorphic “scaffold” oligonucleotides. Top strands were then linked by gap filling and ligation. This approach avoided heteroduplex annealing in the linkage of highly degenerate oligonucleotides and thus achieved completely random recombination. Cloned genes from a human–mouse chimeric library captured every possible permutation of the parental polymorphisms, creating an apparently complete recombined gene-family library, which has not been previously described. This library yielded a chimeric protein whose agonist activity was enhanced 123-fold. A second library from five mammalian EGF homologs possessed the highest reported recombination density (1 crossover per 12.4 bp). The five-homolog library yielded the strongest-binding hEGF variant yet reported. In addition, it contained strongly binding EGF variants with antagonist properties. Our less biased approach to DNA shuffling should be useful for the engineering of a wide variety of proteins.

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

Figure 1: Alignment of the genes encoding human and mouse epidermal growth factor.
Figure 2: Degenerate homoduplex recombination (DHR) strategies.
Figure 3: Chimeric genes and polymorphism representation obtained by DHR.
Figure 4: Functional characterization of EGF21, a super-mitogen from the mouse–human library.
Figure 5: Assessing binding and kinase stimulation of variants from a five-species DHR library.

Similar content being viewed by others

References

  1. Mendelsohn, J. The epidermal growth factor receptor as a target for cancer therapy. Endocr. Relat. Cancer 8, 3–9 (2001).

    Article  CAS  Google Scholar 

  2. Reddy, C.C., Niyogi, S.K., Wells, A., Wiley, H.S. & Lauffenburger, D.A. Engineering epidermal growth factor for enhanced mitogenic potency. Nat. Biotechnol. 14, 1696–1699 (1996).

    Article  CAS  Google Scholar 

  3. Matsunami, R.K., Campion, S.R., Niyogi, S.K. & Stevens, A. Analogs of human epidermal growth factor which partially inhibit the growth factor-dependent protein-tyrosine kinase activity of the epidermal growth factor receptor. FEBS Lett. 264, 105–108 (1990).

    Article  CAS  Google Scholar 

  4. Mullenbach, G.T. et al. Modification of a receptor-binding surface of epidermal growth factor (EGF): analogs with enhanced receptor affinity at low pH or at neutrality. Protein Eng. 11, 473–480 (1998).

    Article  CAS  Google Scholar 

  5. Souriau, C., Gracy, J., Chiche, L. & Weill, M. Direct selection of EGF mutants displayed on filamentous phage using cells overexpressing EGF receptor. Biol. Chem. 380, 451–458 (1999).

    Article  CAS  Google Scholar 

  6. Souriau, C. et al. A simple luciferase assay for signal transduction activity detection of epidermal growth factor displayed on phage. Nucleic Acids Res. 25, 1585–1590 (1997).

    Article  CAS  Google Scholar 

  7. van de Poll, M.L., van Vugt, M.J., Lenferink, A.E. & van Zoelen, E.J. Identification of the minimal requirements for binding to the human epidermal growth factor (EGF) receptor using chimeras of human EGF and an EGF repeat of Drosophila Notch. J. Biol. Chem. 273, 16075–16081 (1998).

    Article  CAS  Google Scholar 

  8. Van Zoelen, E.J., Stortelers, C., Lenferink, A.E. & Van de Poll, M.L. The EGF domain: requirements for binding to receptors of the ErbB family. Vitam. Horm. 59, 99–131 (2000).

    Article  CAS  Google Scholar 

  9. Groenen, L.C., Nice, E.C. & Burgess, A.W. Structure–function relationships for the EGF/TGF-α family of mitogens. Growth Factors 11, 235–257 (1994).

    Article  CAS  Google Scholar 

  10. Pompon, D. & Nicolas, A. Protein engineering by cDNA recombination in yeasts: shuffling of mammalian cytochrome P-450 functions. Gene 83, 15–24 (1989).

    Article  CAS  Google Scholar 

  11. Stemmer, W.P. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 (1994).

    Article  CAS  Google Scholar 

  12. Crameri, A., Raillard, S.A., Bermudez, E. & Stemmer, W.P. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391, 288–291 (1998).

    Article  CAS  Google Scholar 

  13. Coco, W.M. et al. DNA shuffling method for generating highly recombined genes and evolved enzymes. Nat. Biotechnol. 19, 354–359 (2001).

    Article  CAS  Google Scholar 

  14. Gibbs, M.D., Nevalainen, K.M. & Bergquist, P.L. Degenerate oligonucleotide gene shuffling (DOGS): a method for enhancing the frequency of recombination with family shuffling. Gene 271, 13–20 (2001).

    Article  CAS  Google Scholar 

  15. Moore, G.L., Maranas, C.D., Lutz, S. & Benkovic, S.J. Predicting crossover generation in DNA shuffling. Proc. Natl. Acad. Sci. USA 98, 3226–3231 (2001).

    Article  CAS  Google Scholar 

  16. Paabo, S., Irwin, D.M. & Wilson, A.C. DNA damage promotes jumping between templates during enzymatic amplification. J. Biol. Chem. 265, 4718–4721 (1990).

    CAS  PubMed  Google Scholar 

  17. Levichkin, I.V., Shul'ga, A.A., Kurbanov, F.T. & Kirpichnikov, M.P. A new method of designing hybrid genes—the homolog recombination method. Mol. Biol. (Mosk) 29, 983–991 (1995).

    CAS  Google Scholar 

  18. Stemmer, W.P.C. Searching sequence space. Biotechnology 13, 549–553 (1995).

    CAS  Google Scholar 

  19. Stemmer, W.P. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91, 10747–10751 (1994).

    Article  CAS  Google Scholar 

  20. Ostermeier, M., Nixon, A.E., Shim, J.H. & Benkovic, S.J. Combinatorial protein engineering by incremental truncation. Proc. Natl. Acad. Sci. USA 96, 3562–3567 (1999).

    Article  CAS  Google Scholar 

  21. Kikuchi, M., Ohnishi, K. & Harayama, S. An effective family shuffling method using single-stranded DNA. Gene 243, 133–137 (2000).

    Article  CAS  Google Scholar 

  22. Voigt, C.A., Kauffman, S. & Wang, Z.G. Rational evolutionary design: the theory of in vitro protein evolution. Adv. Protein Chem. 55, 79–160 (2001).

    Article  Google Scholar 

  23. Voigt, C.A., Mayo, S.L., Arnold, F.H. & Wang, Z.G. Computational method to reduce the search space for directed protein evolution. Proc. Natl. Acad. Sci. USA 98, 3778–3783 (2001).

    Article  CAS  Google Scholar 

  24. Moore, J.C. & Arnold, F.H. Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents. Nat. Biotechnol. 14, 458–467 (1996).

    Article  CAS  Google Scholar 

  25. Coco, W.M. RACHITT: gene family shuffling by random chimeragenesis on transient templates. Methods Mol. Biol, in press (2003).

  26. van de Poll, M.L., van Vugt, M.J., Lenferink, A.E. & van Zoelen, E.J. Insertion of Argos sequences into the B-loop of epidermal growth factor results in a low-affinity ligand with strong agonistic activity. Biochemistry 36, 7425–7431 (1997).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Beat Blattmann, Steve Palmer, Maureen Downey, Gabe Villarreal, and Chanda Tavoloni for robotic screening assays, competition binding assays, and DNA sequencing.

Author information

Author notes

  1. Wayne M. Coco, Lance P. Encell, A. Katrina Loomis & Nicole Sica

    Present address: Tanox, Inc., Houston, TX, 77025

  2. Michael J. Crist

    Present address: Lexicon Genetics, Inc., The Woodlands, TX, 77381

  3. Laura L. Licato

    Present address: Isolagen, Inc., Houston, TX, 77042

  4. Joseph J. Arensdorf

    Present address: Baker Petrolite, Sugar Land, TX, 77478

  5. Philip T. Pienkos

    Present address: Fermentation Developments, The Woodlands, TX, 77381

  6. Daniel J. Monticello

    Present address: Molecular Logix Inc., 4200 Research Forest Drive, The Woodlands, TX, 77381

Authors and Affiliations

  1. Enchira Biotechnology Corporation, 4200 Research Forest Drive, The Woodlands, 77381, TX

    Wayne M. Coco, Lance P. Encell, William E. Levinson, Michael J. Crist, A. Katrina Loomis, Laura L. Licato, Joseph J. Arensdorf, Nicole Sica, Philip T. Pienkos & Daniel J. Monticello

Authors
  1. Wayne M. Coco
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lance P. Encell
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. William E. Levinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael J. Crist
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. A. Katrina Loomis
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Laura L. Licato
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Joseph J. Arensdorf
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Nicole Sica
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Philip T. Pienkos
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Daniel J. Monticello
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Wayne M. Coco.

Ethics declarations

Competing interests

The work was funded by Enchira Biotechnology Corporation and was completed when the authors were employed by Enchira Biotechnology Corporation. The revised manuscript was submitted after the authors were no longer employed by the company and had no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Coco, W., Encell, L., Levinson, W. et al. Growth factor engineering by degenerate homoduplex gene family recombination. Nat Biotechnol 20, 1246–1250 (2002). https://doi.org/10.1038/nbt757

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt757

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