Journal
NATURE COMMUNICATIONS
Volume 6, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/ncomms9168
Keywords
-
Categories
Funding
- National Institutes of Health [NIDDK-K01DK089006, P01DK01743341]
- Defense Advanced Research Projects Agency [N66001-12-C-4211]
- David and Lucille Packard Foundation Fellowship [2011-37152]
- NIH [T32GM100884]
- National Science Foundation [DGE-1122492]
- US Department of Energy [152339.5055249.100]
- Gen9 Inc.
- DuPont Inc.
- Arnold and Mabel Beckman Foundation
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available