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Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library

Nature Biotechnology volume 21, pages 679–685 (2003)Cite this article

Abstract

This report describes the selection of highly efficient antibody catalysts by combining chemical selection from a synthetic library with directed in vitro protein evolution. Evolution started from a naive antibody library displayed on phage made from fully synthetic, antibody-encoding genes (the Human Combinatorial Antibody Library; HuCAL-scFv). HuCAL-scFv was screened by direct selection for catalytic antibodies exhibiting phosphatase turnover. The substrate used was an aryl phosphate, which is spontaneously transformed into an electrophilic trapping reagent after cleavage. Chemical selection identified an efficient biocatalyst that then served as a template for error-prone PCR (epPCR) to generate randomized repertoires that were subjected to further selection cycles. The resulting superior catalysts displayed cumulative mutations throughout the protein sequence; the ten-fold improvement of their catalytic proficiencies (>1010 M−1) resulted from increased kcat values, thus demonstrating direct selection for turnover. The strategy described here makes the search for new catalysts independent of the immune system and the antibody framework.

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Figure 1: Capturing phage for catalyst selection with a mechanism-based reaction scheme.
Figure 2: Catalyst screening for covalent linkage with the turnover-based substrate.
Figure 3: Catalytic properties of scFv TT1 compared to alkaline phosphatase (AP).
Figure 4: Three-dimensional homology model structure of scFv antibody fragment TT1.
Figure 5: Catalytic properties of TT1 mutants.

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Acknowledgements

S.C.-T. and D.L. were supported by Studentships under the European Commission Training and Mobility of Researchers program (grant ERBFMRXCT 980193). J.R. was supported by a Biotechnology and Biological Sciences Research Council Studentship. We are grateful to Aziz Mekhalfia and Jason Betley for assistance with chemical synthesis. We are indebted to Bernhard Schimmele, Lutz Jermutus, Stephen Marino and Patrik Forrer for help, advice and discussion, and to MorphoSys AG for the constructive collaboration on HuCAL-scFv.

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  1. Dimitrios Lagos

    Present address: Wolfson Institute for Biomedical Research, University College London, Gower Street, WC1E 6BT, London, UK

Authors and Affiliations

  1. Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, Zürich, CH-8057, Switzerland

    Sandro Cesaro-Tadic, Annemarie Honegger & Andreas Plückthun

  2. Department of Molecular Biology Biotechnology, Krebs Institute, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK

    Dimitrios Lagos & Lynda J Partridge

  3. Department of Chemistry Sheffield University, Krebs Institute, Sheffield University, Sheffield, S3 7HF, UK

    James H Rickard & G Michael Blackburn

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Correspondence to G Michael Blackburn or Andreas Plückthun.

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Cesaro-Tadic, S., Lagos, D., Honegger, A. et al. Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library. Nat Biotechnol 21, 679–685 (2003). https://doi.org/10.1038/nbt828

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