Robust production of recombinant phosphoproteins using cell-free protein synthesis

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 () and Michael C. Jewett ()
Additional contact information
Javin P. Oza: Northwestern University
Hans R. Aerni: Yale University
Natasha L. Pirman: Yale University
Karl W. Barber: Yale University
Charlotte M. ter Haar: Northwestern University
Svetlana Rogulina: Yale University
Matthew B. Amrofell: Northwestern University
Farren J. Isaacs: Systems Biology Institute, Yale University
Jesse Rinehart: Yale University
Michael C. Jewett: Northwestern University

Nature Communications, 2015, vol. 6, issue 1, 1-7

Abstract: 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.

Date: 2015
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DOI: 10.1038/ncomms9168

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