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*, Michael C. Jewett

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

82 Scopus citations


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.

Original languageEnglish (US)
Article number8168
JournalNature communications
StatePublished - Sep 9 2015

ASJC Scopus subject areas

  • Chemistry(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Physics and Astronomy(all)


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