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:

In vitro abzyme evolution to optimize antibody recognition for catalysis

Nature Biotechnology volume 19, pages 563–567 (2001)Cite this article

Abstract

Enzymes have evolved their ability to use binding energies for catalysis by increasing the affinity for the transition state of a reaction and decreasing the affinity for the ground state. To evolve abzymes toward higher catalytic activity, we have reconstructed an enzyme-evolutionary process in vitro. Thus, a phage-displayed combinatorial library from a hydrolytic abzyme, 6D9, generated by the conventional in vivo method with immunization of the transition-state analog (TSA), was screened against a newly devised TSA to optimize the differential affinity for the transition state relative to the ground state. The library format successfully afforded evolved variants with 6- to 20-fold increases in activity (kcat) as compared with 6D9. Structural analysis revealed an advantage of the in vitro evolution over the in vivo evolution: an induced catalytic residue in the evolved abzyme arises from double mutations in one codon, which rarely occur in somatic hypermutation in the immune response.

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: Antibody-catalyzed prodrug activation.
Figure 2: Construction of the phage-displayed 6D9 light-chain CDR1 library in the pComb3 vector.
Figure 3: Superposition of the active sites of the parent 6D9 (white) and the variant 8Hf (blue).
Figure 4: Plots of (A) log (Km/Ki) versus log (kcat/kuncat) and (B) log (Ki) versus log (kcat/kuncat) of catalytic antibodies obtained by in vivo (○) and in vitro evolution (●).

Similar content being viewed by others

References

  1. Tramontano, A., Janda, K.D. & Lerner, R.A. Catalytic antibodies. Science 234, 1566–1570 (1986).

    Article  CAS  Google Scholar 

  2. Pollack, S.J., Jacobs, J.W. & Schultz, P.G. Selective chemical catalysis by an antibody. Science 234, 1570–1573 (1986).

    Article  CAS  Google Scholar 

  3. Lerner, R.A., Benkovic, S.J. & Schultz, P.G. At the crossroads of chemistry and immunology: catalytic antibodies. Science 252, 659–667 (1991).

    Article  CAS  Google Scholar 

  4. Schultz, P.G. & Lerner, R.A. From molecular diversity to catalysis: lessons from the immune system. Science 269, 1835–1842 (1995).

    Article  CAS  Google Scholar 

  5. Wirsching, P., Ashley, J.A., Lo, C-H.L., Janda, K.D. & Lerner, R.A. Reactive immunization. Science 270, 1775–1782 (1995).

    Article  CAS  Google Scholar 

  6. Wentworth, P. Jr. et al. A bait and switch hapten strategy generates catalytic antibodies for phosphodiester hydrolysis. Proc. Natl. Acad. Sci. USA 95, 5971–5975 (1998).

    Article  CAS  Google Scholar 

  7. Barbas, C.F., Kang, A.S., Lerner, R.A. & Benkovic, S.J. Assembly of combinatorial antibody libraries on phage surface: the gene III site. Proc. Natl. Acad. Sci. USA 88, 7978–7982 (1991).

    Article  CAS  Google Scholar 

  8. Janda, K.D. et al. Chemical selection for catalysis in combinatorial antibody libraries. Science 275, 945–948 (1997).

    Article  CAS  Google Scholar 

  9. Arkin, M.R. & Wells, J.A. Probing the importance of second sphere residues in an esterolytic antibody by phage display. J. Mol. Biol. 284, 1083–1094 (1998).

    Article  CAS  Google Scholar 

  10. Gao, G. et al. Direct selection for catalysis from combinatorial antibody libraries using a boronic acid probe: primary amide bond hydrolysis. J. Am. Chem. Soc. 120, 2211–2217 (1998).

    Article  CAS  Google Scholar 

  11. Fujii, I. Fukuyama, S., Iwabuchi, Y. & Tanimura, R. Evolving catalytic antibodies in a phage-displayed combinatorial library. Nat. Biotechnol. 16, 463–467 (1998).

    Article  CAS  Google Scholar 

  12. Tanaka, F., Lerner, R.A. & Barbas, C.F. Reconstructing aldolase antibodies to alter their substrate specificity and turnover. J. Am. Chem. Soc. 122, 4835–4836 (2000).

    Article  CAS  Google Scholar 

  13. Ulrich, H.D. et al. The interplay between binding energy and catalysis in the evolution of a catalytic antibody. Nature 389, 271–275 (1997).

    Article  CAS  Google Scholar 

  14. Tonegawa, S. Somatic generation of antibody diversity. Nature 302, 575–581 (1983).

    Article  CAS  Google Scholar 

  15. Neuberger, M. & Milstein, C. Somatic hypermutation. Curr. Opin. Immunol. 7, 248–254 (1995).

    Article  CAS  Google Scholar 

  16. Pauling, L. Chemical achievement and hope for the future. Am. Sci. 36, 51–58 (1948).

    CAS  PubMed  Google Scholar 

  17. Lienhard, G.E. Enzymatic catalysis and transition-state theory. Science 180, 149–154 (1973).

    Article  CAS  Google Scholar 

  18. Fersht, A. Enzyme structure and mechanism, Edn. 2 (W.H. Freeman and Co., New York, NY; 1985).

    Google Scholar 

  19. Miyashita, H., Karaki, Y., Kikuchi, M. & Fujii, I. Prodrug activation via catalytic antibodies. Proc. Natl. Acad. Sci. USA 90, 5337–5340 (1993).

    Article  CAS  Google Scholar 

  20. Miyashita. H. et al. A common ancestry for multiple catalytic antibodies generated against a single transition-state analog. Proc. Natl. Acad. Sci. USA 91, 6045–6049 (1994).

    Article  CAS  Google Scholar 

  21. Fujii, I., Tanaka, F., Miyashita, H., Tanimura, R. & Kinoshita, K. Correlation between antigen-combining-site structures and functions within a panel of catalytic antibodies generated against a single transition state analog. J. Am. Chem. Soc. 117, 6199–6209 (1995).

    Article  CAS  Google Scholar 

  22. Kabat, E.A., Wu, T.T., Perry, H.M., Gottesman, K.S. & Foeller, C. Sequences of proteins of immunological interest, Edn. 5. (US Department of Health and Human Services. National Institutes of Health, Bethesda, MD; 1991).

    Google Scholar 

  23. Miyashita, H. et al. Site-directed mutagenesis of active site contact residues in a hydrolytic abzyme: evidence for an essential histidine involved in transition state stabilization. J. Mol. Biol. 267, 1247–1257 (1997).

    Article  CAS  Google Scholar 

  24. Kristensen, O., Vassylyev, D.G., Tanaka, F., Morikawa, K. & Fujii, I. A structural basis for transition-state stabilization in antibody-catalyzed hydrolysis: crystal structures of an abzyme at 1.8 Å resolution. J. Mol. Biol. 281, 501–511 (1998).

    Article  CAS  Google Scholar 

  25. Cagnon, C., Valverde, V. & Masson, J.-M. A new family of sugar-inducible expression vectors for Escherichia coli. Protein Eng. 4, 843–847 (1991).

    Article  CAS  Google Scholar 

  26. Fersht, A.R. et al. Hydrogen bonding and biological specificity analysed by protein engineering. Nature 314, 235–238 (1985).

    Article  CAS  Google Scholar 

  27. Corbet, S., Milili, M., Fougereau, M. & Schiff, C. Two Vκ germ-line genes related to the GAT idiotypic network (Ab1 and Ab3/Ab1') account for the major subfamilies of the mouse Vκ-1 variability subgroup. J. Immunol. 138, 932–939 (1987).

    CAS  PubMed  Google Scholar 

  28. Huse, W.D. et al. Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 246, 1275–1281 (1989).

    Article  CAS  Google Scholar 

  29. Takahashi, N. et al. Efficient screening for catalytic antibodies using a short transition-state analog and detailed characterization of selected antibodies. Eur. J. Biochem. 261, 108–114 (1999).

    Article  CAS  Google Scholar 

  30. Angeles, T.S. et al. Isoabzymes: structurally and mechanistically similar catalytic antibodies from the same immunization. Biochemistry 32, 12128–12135 (1993).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Ms. K. Inoue for her technical assistance and Dr. K. Akiyama, Dr. Y. Aoki, and Dr. K. Shimazaki for their helpful suggestions. This research was supported by the New Energy and Industrial Technology Development Organization as an R&D project of the Industrial Science and Technology Frontier Program.

Author information

Authors and Affiliations

  1. Laboratory of Life Science and Biomolecular Engineering, Japan Tobacco, Inc., 6-2, Umegaoka, Aoba-ku, Yokohama, 227-8512, Kanagawa, Japan

    Naoko Takahashi, Hiroyuki Kakinuma & Yoshisuke Nishi

  2. Biomolecular Engineering Research Institute, 6-2-3, Furuedai, Suita, 565-0874, Osaka, Japan

    Lidong Liu & Ikuo Fujii

Authors
  1. Naoko Takahashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hiroyuki Kakinuma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lidong Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yoshisuke Nishi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ikuo Fujii
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ikuo Fujii.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Takahashi, N., Kakinuma, H., Liu, L. et al. In vitro abzyme evolution to optimize antibody recognition for catalysis. Nat Biotechnol 19, 563–567 (2001). https://doi.org/10.1038/89320

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/89320

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