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Robust production of recombinant phosphoproteins using cell-free protein synthesis

Author

Listed:
  • Javin P. Oza

    (Northwestern University
    Northwestern Institute on Complex Systems, Northwestern University
    Simpson Querrey Institute, Northwestern University
    Chemistry of Life Processes Institute, Northwestern University)

  • Hans R. Aerni

    (Yale University
    Systems Biology Institute, Yale University)

  • Natasha L. Pirman

    (Yale University
    Systems Biology Institute, Yale University)

  • Karl W. Barber

    (Yale University
    Systems Biology Institute, Yale University)

  • Charlotte M. ter Haar

    (Northwestern University)

  • Svetlana Rogulina

    (Yale University
    Systems Biology Institute, Yale University)

  • Matthew B. Amrofell

    (Northwestern University)

  • Farren J. Isaacs

    (Systems Biology Institute, Yale University
    Cellular and Developmental Biology, Yale University)

  • Jesse Rinehart

    (Yale University
    Systems Biology Institute, Yale University)

  • Michael C. Jewett

    (Northwestern University
    Northwestern Institute on Complex Systems, Northwestern University
    Simpson Querrey Institute, Northwestern University
    Chemistry of Life Processes Institute, Northwestern University)

Abstract

Understanding the functional and structural consequences of site-specific protein phosphorylation has remained limited by our inability to produce phosphoproteins at high yields. Here we address this limitation by developing a cell-free protein synthesis (CFPS) platform that employs crude extracts from a genomically recoded strain of Escherichia coli for site-specific, co-translational incorporation of phosphoserine into proteins. We apply this system to the robust production of up to milligram quantities of human MEK1 kinase. Then, we recapitulate a physiological signalling cascade in vitro to evaluate the contributions of site-specific phosphorylation of mono- and doubly phosphorylated forms on MEK1 activity. We discover that only one phosphorylation event is necessary and sufficient for MEK1 activity. Our work sets the stage for using CFPS as a rapid high-throughput technology platform for direct expression of programmable phosphoproteins containing multiple phosphorylated residues. This work will facilitate study of phosphorylation-dependent structure–function relationships, kinase signalling networks and kinase inhibitor drugs.

Suggested Citation

  • Javin P. Oza & Hans R. Aerni & Natasha L. Pirman & Karl W. Barber & Charlotte M. ter Haar & Svetlana Rogulina & Matthew B. Amrofell & Farren J. Isaacs & Jesse Rinehart & Michael C. Jewett, 2015. "Robust production of recombinant phosphoproteins using cell-free protein synthesis," Nature Communications, Nature, vol. 6(1), pages 1-7, November.
  • Handle: RePEc:nat:natcom:v:6:y:2015:i:1:d:10.1038_ncomms9168
    DOI: 10.1038/ncomms9168
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    Cited by:

    1. Remkes A. Scheele & Laurens H. Lindenburg & Maya Petek & Markus Schober & Kevin N. Dalby & Florian Hollfelder, 2022. "Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates," Nature Communications, Nature, vol. 13(1), pages 1-14, December.

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