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Measuring the activity of protein variants on a large scale using deep mutational scanning

Nature Protocols volume 9, pages 2267–2284 (2014)Cite this article

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Abstract

Deep mutational scanning marries selection for protein function to high-throughput DNA sequencing in order to quantify the activity of variants of a protein on a massive scale. First, an appropriate selection system for the protein function of interest is identified and validated. Second, a library of variants is created, introduced into the selection system and subjected to selection. Third, library DNA is recovered throughout the selection and deep-sequenced. Finally, a functional score for each variant is calculated on the basis of the change in the frequency of the variant during the selection. This protocol describes the steps that must be carried out to generate a large-scale mutagenesis data set consisting of functional scores for up to hundreds of thousands of variants of a protein of interest. Establishing an assay, generating a library of variants and carrying out a selection and its accompanying sequencing takes on the order of 4–6 weeks; the initial data analysis can be completed in 1 week.

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Figure 1: Deep mutational scanning workflow.
Figure 2: Variable library sequencing methods.
Figure 3: Using the Enrich software to analyze deep mutational scanning data.
Figure 4: Creating a barcoded library from Gibson-assembled oligonucleotides.

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Acknowledgements

This work was supported by grants P41 GM103533 (to S.F.) and F32 GM084699 (to D.M.F.) from the US National Institute of General Medical Sciences. S.F. is supported by the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Genome Sciences, University of Washington, Seattle, Washington, USA

    Douglas M Fowler, Jason J Stephany & Stanley Fields

  2. Department of Medicine, University of Washington, Seattle, Washington, USA

    Stanley Fields

  3. Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA

    Stanley Fields

Authors
  1. Douglas M Fowler
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  2. Jason J Stephany
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  3. Stanley Fields
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Contributions

D.M.F. and J.J.S. developed and refined the protocols; and D.M.F., J.J.S. and S.F. wrote the paper.

Corresponding author

Correspondence to Douglas M Fowler.

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The authors declare no competing financial interests.

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Fowler, D., Stephany, J. & Fields, S. Measuring the activity of protein variants on a large scale using deep mutational scanning. Nat Protoc 9, 2267–2284 (2014). https://doi.org/10.1038/nprot.2014.153

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