High-resolution sequence-function mapping of full-length proteins

Caitlin A. Kowalsky, Justin R. Klesmith, James A. Stapleton, Vince Kelly, Nolan Reichkitzer, Timothy A. Whitehead

    Research output: Research - peer-reviewArticle

    • 13 Citations

    Abstract

    Comprehensive sequence-function mapping involves detailing the fitness contribution of every possible single mutation to a gene by comparing the abundance of each library variant before and after selection for the phenotype of interest. Deep sequencing of library DNA allows frequency reconstruction for tens of thousands of variants in a single experiment, yet short read lengths of current sequencers makes it challenging to probe genes encoding fulllength proteins. Here we extend the scope of sequence-function maps to entire protein sequences with a modular, universal sequence tiling method. We demonstrate the approach with both growth-based selections and FACS screening, offer parameters and best practices that simplify design of experiments, and present analytical solutions to normalize data across independent selections. Using this protocol, sequence-function maps covering full sequences can be obtained in four to six weeks. Best practices introduced in this manuscript are fully compatible with, and complementary to, other recently published sequencefunction mapping protocols.

    LanguageEnglish (US)
    Article numbere0118193
    JournalPLoS One
    Volume10
    Issue number3
    DOIs
    StatePublished - Mar 19 2015

    Profile

    genes
    proteins
    Practice Guidelines
    Genes
    Proteins
    DNA libraries
    amino acid sequences
    experimental design
    screening
    mutation
    phenotype
    DNA
    methodology
    high-throughput nucleotide sequencing
    High-Throughput Nucleotide Sequencing
    Gene Library
    Libraries
    Phenotype
    Mutation
    Growth

    ASJC Scopus subject areas

    • Agricultural and Biological Sciences(all)
    • Biochemistry, Genetics and Molecular Biology(all)
    • Medicine(all)

    Cite this

    Kowalsky, C. A., Klesmith, J. R., Stapleton, J. A., Kelly, V., Reichkitzer, N., & Whitehead, T. A. (2015). High-resolution sequence-function mapping of full-length proteins. PLoS One, 10(3), [e0118193]. DOI: 10.1371/journal.pone.0118193

    High-resolution sequence-function mapping of full-length proteins. / Kowalsky, Caitlin A.; Klesmith, Justin R.; Stapleton, James A.; Kelly, Vince; Reichkitzer, Nolan; Whitehead, Timothy A.

    In: PLoS One, Vol. 10, No. 3, e0118193, 19.03.2015.

    Research output: Research - peer-reviewArticle

    Kowalsky, CA, Klesmith, JR, Stapleton, JA, Kelly, V, Reichkitzer, N & Whitehead, TA 2015, 'High-resolution sequence-function mapping of full-length proteins' PLoS One, vol 10, no. 3, e0118193. DOI: 10.1371/journal.pone.0118193
    Kowalsky CA, Klesmith JR, Stapleton JA, Kelly V, Reichkitzer N, Whitehead TA. High-resolution sequence-function mapping of full-length proteins. PLoS One. 2015 Mar 19;10(3). e0118193. Available from, DOI: 10.1371/journal.pone.0118193
    Kowalsky, Caitlin A. ; Klesmith, Justin R. ; Stapleton, James A. ; Kelly, Vince ; Reichkitzer, Nolan ; Whitehead, Timothy A./ High-resolution sequence-function mapping of full-length proteins. In: PLoS One. 2015 ; Vol. 10, No. 3.
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