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Enzyme Engineering pp 205–216Cite as

Promiscuity-Based Enzyme Selection for Rational Directed Evolution Experiments

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 978))

Abstract

Error-prone PCR, DNA shuffling, and saturation mutagenesis are techniques used by protein engineers to mimic the natural “evolutionary walk” that conjures new enzymes. Rational design is often critical in efforts to accelerate this “random walk” into a “resolute sprint.” Previous work by our group established a computational method for detecting active sites (CLASP) based on spatial and electrostatic properties of catalytic residues, and a method to quantify promiscuous activities in a wide range of proteins (PROMISE). Here, we describe a rational design flow (DECAAF) based on the PROMISE methodology to choose a protein which, when subjected to minimal mutations, is most likely to mirror the scaffold of a desired enzymatic function. Modeling the diversity in catalytic sites and providing precise user control to guide the search is a key goal of our implementation. The flow details have been worked out in a real-life example to select a plant protein to substitute for human neutrophil elastase in a chimeric antimicrobial enzyme designed to bolster the innate immune defense system in plants.

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Acknowledgements

We are deeply indebted to J. M. Frere (Centre for Protein Engineering, Universite de Liege, Institut de Chimie B6, Sart Tilman, B-4000 Liege, Belgium), Bjarni Asgeirsson (Science Institute, Department of Biochemistry, University of Iceland), Masataka Oda (Department of Microbiology, Faculty of Pharmaceutical Science, Tokushima Bunri University, Japan), and Felix M. Goni (Unidad de Biofisica (CSIC-UPV/EHU) and Departamento de Bioquimica, Universidad del Pais Vasco, Bilbao, Spain), for technical discussions, suggestions, and support. AMD acknowledges support received from the California Department of Food and Agriculture’s Pierce’s Disease Board to conduct this collaborative research. BJR acknowledges a J.C. Bose award fellowship grant.

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Authors and Affiliations

  1. Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India

    Sandeep Chakraborty, Renu Minda, Lipika Salaye, Swapan K. Bhattacharjee & Basuthkar J. Rao

  2. Plant Sciences Department, University of California, Davis, CA, USA

    Abhaya M. Dandekar

Authors
  1. Sandeep Chakraborty
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  2. Renu Minda
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  3. Lipika Salaye
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  4. Abhaya M. Dandekar
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  5. Swapan K. Bhattacharjee
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  6. Basuthkar J. Rao
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Correspondence to Sandeep Chakraborty .

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Editors and Affiliations

  1. New England Biolabs, Inc., County Road 240, Ipswich, 01938, Massachusetts, USA

    James C. Samuelson

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Chakraborty, S., Minda, R., Salaye, L., Dandekar, A.M., Bhattacharjee, S.K., Rao, B.J. (2013). Promiscuity-Based Enzyme Selection for Rational Directed Evolution Experiments. In: Samuelson, J. (eds) Enzyme Engineering. Methods in Molecular Biology, vol 978. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-293-3_15

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  • DOI: https://doi.org/10.1007/978-1-62703-293-3_15

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  • Publisher Name: Humana Press, Totowa, NJ

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