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.

Kan R plasmids containing SupD or SepOTSλ were tested with a dual-marker plasmid harboring a TAG-containing beta-lactamase gene (S36TAG) and a chloramphenicol acetyltransferase cassette. (c) Total beta-lactamase (S36TAG) expression confirmed by western blot. Expression of total and phospho-MBP-MEK1 is compared across BL21 RF1+, EcAR7.ΔA, and C321.ΔA strains using SepOTSμ. Loading was normalized by OD 600 measurement.   Table 1) however it was not possible to observe the doubly phosphorylated peptide presumably due to poor ionization. We therefore devised an           The two SepOTSα plasmids were previously described as pSepT and pKD-SepRS-EFSep

Northern blot
Cell pellets corresponding to 1 mL of OD 600 2.5 of cells were resuspended in 0.5 mL of RNA extraction buffer (0.3 M sodium acetate, 10 mM EDTA, pH 4.5). 0.5 mL of acidic phenol was added to each tube, and samples were vortexed for 10 sec. Samples were then incubated on ice for 15 min, with quick vortexing every 3 min. Samples were then centrifuged for 12 min at 12,000 x g at 4 °C. The aqueous phase was transferred to a new tube, and 0.25 mL of RNA extraction buffer was added to each tube, vortexed, and centrifuged again. The aqueous phases were combined. 700 μL of ethanol was added to each tube, and tubes were frozen at -80 °C overnight. Samples were then spun at 13,000 x g for 20 min at 4 °C and supernatant was removed. Pellets were washed with 1 mL ice cold 70% ethanol and centrifuged at 13,000 x g for 12 min at 4 °C. Ethanol was removed and pellets were allowed to dry.  6 . Images were collected using a Typhoon FLA9000 and densitometry was performed using ImageQuant TL v7.0 (GE Healthcare).

S36TAG beta-lactamase plate assays
C321.ΔA cells containing the pCRT7 NT Topo tetR pLtetO plasmid including betalactamase S36TAG and either SepOTSλ or the SupD plasmid were grown from glycerol stocks, induced, and grown for 20 hrs as for E17TAG GFP protein expression above. Cultures were diluted to OD 600 of 2.0AU in LB media and 2 μL of cells were spotted on LB agar plates with either kanamycin (25 μg per mL) and chloramphenicol (10 μg per mL) or kanamycin (25 μg per mL) and ampicillin (100 μg per mL). Plates were grown ~20 hrs at 30 °C and images were acquired with a Bio-Rad Gel Doc imager.
The same cultures used for the plate assays were run on a 4-15% acrylamide SDS-PAGE gel

Growth curves
EcAR7.ΔA and C321.ΔA strains containing the MBP-MEK1 (S218TAG/S222TAG) and SepOTSμ plasmids were freshly streaked from glycerol stocks on LB agar plates supplemented with ampicillin (100 μg per mL), kanamycin (25 μg per mL) and 0.08% glucose. 5 mL of selective LB media supplemented with 0.08% glucose was inoculated with 5 colonies and grown to confluency. The precultures were back diluted to OD 600 of 0.8 AU then diluted 1:100 into a 96-well cell culture plate (Costar 3595, Corning) containing LB media with 0.08% glucose, antibiotics, 2 mM Sep, 100 ng per mL ATC, and 1 mM IPTG in 5x replicates for each culture.
The plate was incubated and continuously shaken (medium speed) at 30 °C in a Biotek Synergy HT plate reader monitoring OD 600 in 10 min intervals for 24 hrs.

MBP-MEK1 treatment with calf intestinal alkaline phosphatase (CIP)
Affinity-purified MBP-MEK1 S P S P protein (45 µg total protein) was treated with calf intestinal alkaline phosphatase (CIP, NEB) using 45 units of enzyme with 1x NEB buffer 3 in a total reaction volume of 50 µL. The dephosphorylation reaction was carried out at 37 °C for 1 hr.
Samples were then dialyzed twice against 1 L cold 10 mM Tris-HCl buffer pH 8.0 using Slide-A-Lyzer TM MINI devices with 2 kDa molecular weight cutoff (Life Technologies).

Protein digestion and mass spectrometry
Mass Maisch GmBH). Methanol was used as the packing solvent. Eluent A and Eluent B were 0.1% formic acid and 0.1% formic acid in acetonitrile respectively. The injection volume was 3 µl.