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Nat Commun. 2015 Sep 9;6:8168. doi: 10.1038/ncomms9168.

Robust production of recombinant phosphoproteins using cell-free protein synthesis.

Author information

1
Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.
2
Northwestern Institute on Complex Systems, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.
3
Simpson Querrey Institute, Northwestern University, Chicago, Illinois 60611, USA.
4
Chemistry of Life Processes Institute, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.
5
Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520, USA.
6
Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA.
7
Department of Biomedical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.
8
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06510, USA.
9
Interdisciplinary Biological Sciences Program, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.

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.

PMID:
26350765
PMCID:
PMC4566161
DOI:
10.1038/ncomms9168
[Indexed for MEDLINE]
Free PMC Article
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