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Using the Evolutionary History of Proteins to Engineer Insertion-Deletion Mutants from Robust, Ancestral Templates Using Graphical Representation of Ancestral Sequence Predictions (GRASP)

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Enzyme Engineering

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

Abstract

Analyzing the natural evolution of proteins by ancestral sequence reconstruction (ASR) can provide valuable information about the changes in sequence and structure that drive the development of novel protein functions. However, ASR has also been used as a protein engineering tool, as it often generates thermostable proteins which can serve as robust and evolvable templates for enzyme engineering. Importantly, ASR has the potential to provide an insight into the history of insertions and deletions that have occurred in the evolution of a protein family. Indels are strongly associated with functional change during enzyme evolution and represent a largely unexplored source of genetic diversity for designing proteins with novel or improved properties. Current ASR methods differ in the way they handle indels; inclusion or exclusion of indels is often managed subjectively, based on assumptions the user makes about the likelihood of each recombination event, yet most currently available ASR tools provide limited, if any, opportunities for evaluating indel placement in a reconstructed sequence. Graphical Representation of Ancestral Sequence Predictions (GRASP) is an ASR tool that maps indel evolution throughout a reconstruction and enables the evaluation of indel variants. This chapter provides a general protocol for performing a reconstruction using GRASP and using the results to create indel variants. The method addresses protein template selection, sequence curation, alignment refinement, tree building, ancestor reconstruction, evaluation of indel variants and approaches to library development.

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Author information

Authors and Affiliations

  1. School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD, Australia

    Connie M. Ross, Gabriel Foley, Mikael Boden & Elizabeth M. J. Gillam

Authors
  1. Connie M. Ross
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  2. Gabriel Foley
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  3. Mikael Boden
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  4. Elizabeth M. J. Gillam
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Corresponding author

Correspondence to Elizabeth M. J. Gillam .

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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Ross, C.M., Foley, G., Boden, M., Gillam, E.M.J. (2022). Using the Evolutionary History of Proteins to Engineer Insertion-Deletion Mutants from Robust, Ancestral Templates Using Graphical Representation of Ancestral Sequence Predictions (GRASP). 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_6

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  • DOI: https://doi.org/10.1007/978-1-0716-1826-4_6

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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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