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

By Douglas M. Fowler, Jason J. Stephany, Stanley Fields. 2014

184 citations recorded by the source corpus.

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.

Subfields: Genetics, Molecular Biology

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Citations in the corpus

Quantitative missense variant effect prediction using large-scale mutagenesis data (2017, neutral): “In a deep mutational scan, selection for protein function among a library of nearly all possible single amino acid variants of a protein is coupled to high-throughput DNA sequencing (Fowler and Fields, 2014; Fowler et al., 2014).”
The genetic landscape of a physical interaction (2018, methodology): “Following the recommendations of Fowler et al. (2014), we targeted at least 10/flow » 10 7 bacterial and yeast transformants.”
Multiplex Assessment of Protein Variant Abundance by Massively Parallel Sequencing (2018, background): “PTEN mediates the removal of the 3 ′ phosphate from phosphatidylinositol 3,4,5-triphosphate (PIP 3 ) to produce phosphatidylinositol 4,5-diphosphate (PIP 2 ) at the membrane 28 .”