Robust production of recombinant phosphoproteins using cell-free protein synthesis.
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.
Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.|Northwestern Institute on Complex Systems, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.|Simpson Querrey Institute, Northwestern University, Chicago, Illinois 60611, USA.|Chemistry of Life Processes Institute, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.
Javin P
JP
Oza
Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520, USA.|Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA.
Hans R
HR
Aerni
Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520, USA.|Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA.
Natasha L
NL
Pirman
Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520, USA.|Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA.
Karl W
KW
Barber
Department of Biomedical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.
Charlotte M
CM
Ter Haar
Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520, USA.|Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA.
Svetlana
S
Rogulina
Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.
Matthew B
MB
Amrofell
Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA.|Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06510, USA.
Farren J
FJ
Isaacs
Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520, USA.|Systems Biology Institute, Yale University, West Haven, Connecticut 06516, USA.
Jesse
J
Rinehart
Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.|Northwestern Institute on Complex Systems, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.|Simpson Querrey Institute, Northwestern University, Chicago, Illinois 60611, USA.|Chemistry of Life Processes Institute, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.|Interdisciplinary Biological Sciences Program, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, USA.
Michael C
MC
Jewett
D015153:Blotting, Western; D002474:Cell-Free System; D057075:Enzyme Assays; D004926:Escherichia coli; D049452:Green Fluorescent Proteins; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000096:biosynthesis* / Q000378:metabolism; D013058:Mass Spectrometry; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism
D010750:Phosphoproteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D010768:Phosphoserine; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human