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Recombination of Compatible Substitutions by 2GenReP and InSiReP

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Enzyme Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2397))

Abstract

The CompassR rule enables to identify the beneficial substitutions, which can be recombined in directed evolution with gradually improving the enzymatic properties. However, the question of how to efficiently explore the protein sequence space when ten or more beneficial substitutions are identified has not yet been addressed. Two recombination strategies 2GenReP and InSiReP employing CompassR are systematically investigated to minimize experimental efforts and maximize possible improvements. Here we describe the details of the 2GenReP and InSiReP procedure with an example of recombining 15 substitutions and discuss some important practical issues that should be considered for the application of 2GenReP and InSiReP, such as placing the substitutions into subsets. The core part of the protocol (Step1 to Step5) is transferable to other enzymes and any recombination of potential substitutions.

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Authors and Affiliations

  1. Institute of Biotechnology, RWTH Aachen University, Aachen, Germany

    Haiyang Cui, Mehdi D. Davari & Ulrich Schwaneberg

  2. DWI Leibniz-Institute for Interactive Materials, Aachen, Germany

    Ulrich Schwaneberg

Authors
  1. Haiyang Cui
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  2. Mehdi D. Davari
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  3. Ulrich Schwaneberg
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Corresponding authors

Correspondence to Mehdi D. Davari or Ulrich Schwaneberg .

Editor information

Editors and Affiliations

  1. Department of Biology and Biotechnology, University of Pavia, Pavia, Italy

    Francesca Magnani

  2. Department of Biology and Biotechnology, University of Pavia, Pavia, Italy

    Chiara Marabelli

  3. Department of Chemistry & Biochemistry, University of Bern, Bern, Switzerland

    Francesca Paradisi

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Cui, H., Davari, M.D., Schwaneberg, U. (2022). Recombination of Compatible Substitutions by 2GenReP and InSiReP. In: Magnani, F., Marabelli, C., Paradisi, F. (eds) Enzyme Engineering. Methods in Molecular Biology, vol 2397. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1826-4_5

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  • DOI: https://doi.org/10.1007/978-1-0716-1826-4_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1825-7

  • Online ISBN: 978-1-0716-1826-4

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