A flexible codon in genomically recoded Escherichia coli permits programmable protein phosphorylation.
Biochemical investigation of protein phosphorylation events is limited by inefficient production of the phosphorylated and non-phosphorylated forms of full-length proteins. Here using a genomically recoded strain of E. coli with a flexible UAG codon we produce site-specific serine- or phosphoserine-containing proteins, with purities approaching 90%, from a single recombinant DNA. Specifically, we synthesize human MEK1 kinase with two serines or two phosphoserines, from one DNA template, and demonstrate programmable kinase activity. Programmable protein phosphorylation is poised to help reveal the structural and functional information encoded in the phosphoproteome.
Department of Cellular &Molecular Physiology, Yale University, New Haven, Connecticut 06520-8114, USA.|Systems Biology Institute, Yale University, New Haven, Connecticut 06520-8114, USA.
Natasha L
NL
Pirman
Department of Cellular &Molecular Physiology, Yale University, New Haven, Connecticut 06520-8114, USA.|Systems Biology Institute, Yale University, New Haven, Connecticut 06520-8114, USA.
Karl W
KW
Barber
Department of Cellular &Molecular Physiology, Yale University, New Haven, Connecticut 06520-8114, USA.|Systems Biology Institute, Yale University, New Haven, Connecticut 06520-8114, USA.
Hans R
HR
Aerni
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8114, USA.|Systems Biology Institute, Yale University, New Haven, Connecticut 06520-8114, USA.
Natalie J
NJ
Ma
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8114, USA.|Systems Biology Institute, Yale University, New Haven, Connecticut 06520-8114, USA.
Adrian D
AD
Haimovich
Department of Cellular &Molecular Physiology, Yale University, New Haven, Connecticut 06520-8114, USA.|Systems Biology Institute, Yale University, New Haven, Connecticut 06520-8114, USA.
Svetlana
S
Rogulina
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8114, USA.|Systems Biology Institute, Yale University, New Haven, Connecticut 06520-8114, USA.
Farren J
FJ
Isaacs
Department of Cellular &Molecular Physiology, Yale University, New Haven, Connecticut 06520-8114, USA.|Systems Biology Institute, Yale University, New Haven, Connecticut 06520-8114, USA.
Jesse
J
Rinehart
D018388:Codon, Terminator / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D016680:Genome, Bacterial / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000096:biosynthesis / Q000235:genetics*; D030781:Organisms, Genetically Modified; D010766:Phosphorylation / Q000235:genetics*; D010768:Phosphoserine; D012694:Serine
D018388:Codon, Terminator; D049452:Green Fluorescent Proteins; D010768:Phosphoserine; D012694:Serine; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human