Phage-assisted continuous evolution of proteases with altered substrate specificity View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2017-12

AUTHORS

Michael S. Packer, Holly A. Rees, David R. Liu

ABSTRACT

Here we perform phage-assisted continuous evolution (PACE) of TEV protease, which canonically cleaves ENLYFQS, to cleave a very different target sequence, HPLVGHM, that is present in human IL-23. A protease emerging from ∼2500 generations of PACE contains 20 non-silent mutations, cleaves human IL-23 at the target peptide bond, and when pre-mixed with IL-23 in primary cultures of murine splenocytes inhibits IL-23-mediated immune signaling. We characterize the substrate specificity of this evolved enzyme, revealing shifted and broadened specificity changes at the six positions in which the target amino acid sequence differed. Mutational dissection and additional protease specificity profiling reveal the molecular basis of some of these changes. This work establishes the capability of changing the substrate specificity of a protease at many positions in a practical time scale and provides a foundation for the development of custom proteases that catalytically alter or destroy target proteins for biotechnological and therapeutic applications.Proteases are promising therapeutics to treat diseases such as hemophilia which are due to endogenous protease deficiency. Here the authors use phage-assisted continuous evolution to evolve a variant TEV protease with altered target peptide sequence specificities. More... »

PAGES

956

References to SciGraph publications

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  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/s41467-017-01055-9

    DOI

    http://dx.doi.org/10.1038/s41467-017-01055-9

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1092152760

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/29038472


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