Robust production of recombinant phosphoproteins using cell-free protein synthesis

• Published in: Nature communications 2015-09, Vol.6 (1), p.8168-8168, Article 8168
• Main Authors: Oza, Javin P., Aerni, Hans R., Pirman, Natasha L., Barber, Karl W., ter Haar, Charlotte M., Rogulina, Svetlana, Amrofell, Matthew B., Isaacs, Farren J., Rinehart, Jesse, Jewett, Michael C.
• Format: Article
• Language: English
• Subjects: 38/23; 38/35; 38/70; 631/1647/338; 631/45/612/1246; 82/1; 82/29; 82/58; 82/80; 82/83; 96/35; 96/95; BASIC BIOLOGICAL SCIENCES; Blotting, Western; Cell-Free System; E coli; Enzyme Assays; Enzyme inhibitors; Escherichia coli; Green Fluorescent Proteins; Humanities and Social Sciences; Humans; Kinases; MAP Kinase Kinase 1 - biosynthesis; MAP Kinase Kinase 1 - metabolism; Mass Spectrometry; MEK1 kinase; multidisciplinary; Phosphoproteins; Phosphoproteins - biosynthesis; Phosphoproteins - metabolism; Phosphorylation; Phosphoserine; Phosphoserine - metabolism; Protein Biosynthesis; Protein synthesis; Proteins; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; Science; Science & Technology - Other Topics; Science (multidisciplinary); Signaling; Structure-function relationships
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/ncomms9168

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. The inability to produce recombinant phosphoproteins has hindered research into their structure and function. Here the authors develop a cell-free protein synthesis platform to site-specifically incorporate phosphoserine into proteins at high yields, and recapitulate a MEK1 kinase signalling cascade.