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Rinehart-icon.jpg Rinehart Lab Phosphoprotein Reagents


Phosphoprotein-synthesis

Protein phosphorylation is one of the most abundant forms of posttranslational modifications in cells and research into its many roles in protein function and signaling networks continues to expand. The Rinehart and Söll labs at Yale University changed the way researchers can explore important questions surrounding serine phosphorylation by adding this phosphorylated amino acid to the genetic code of E. coli (Park et al., Science 2011). The Rinehart lab has now made improvements to this system by genomically recoding E. coli (Lajoie et al., Science 2014), and improving the orthogonal translation system to make singly or multiply phosphorylated proteins (Pirman et al., Nat Comm 2015). To demonstrate the improvements of the system they synthesized the activated form of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two phosphoserine residues cotranslationally inserted in their canonical positions (SP218, SP222).

Improved expression of phosphorylated proteins in genomically recoded E. coli . Taking advantage of a recoded strain of E. coli containing no genomic TAG codons (Lajoie et al., Science 2014) and a RF-1 deletion (68306), the Rinehart lab used an E17TAG GFP reporter protein (68295) to empirically determine the best combination of SepOTS components to produce recombinant phosphoproteins at higher yields and purities (Pirman et al., Nat Comm 2015). All of the SepOTS variants investigated in this paper are available on Addgene, but for most routine purposes, SepOTSλ (68292) transformed into the specialized recoded strain (68306) is recommended for robust phosphoprotein synthesis.

They also utilized a serine suppressor tRNA known as SupD (68307) to encode serine at amber codons, allowing incorporation of either serine or phosphoserine and programmable protein activity using a single TAG-containing plasmid. A paper describing cell-free phosphoprotein synthesis making use of some improved SepOTS variants and genomically recoded E. coli may also be of interest to labs attempting site-directed phosphoserine incorporation in recombinant proteins (Oza et al., Nat Comm 2015). Finally, a recent paper describes synthesis of phosphorylated forms of the ubiquitin machinery utilizing SepOTSλ (68292) and the recoded strain (68306 ; Heo, et al., Mol. Cell 2015).

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The synthetic human phosphoproteome. The Rinehart lab synthesized 110,139 unique DNA sequences corresponding to every previously observed instance of human serine phosphorylation (Rinehart Human Serine Phosphopeptide Library - Available Soon). The phosphorylation sites (“phosphosites”) consist of a central phosphoserine site flanked by 15 amino acids on either side occurring within the parent protein. Using the recoded strain of E. coli (68306) with the optimized phosphoserine orthogonal translation system (68292), they were able to demonstrate unambiguous site-specific incorporation of phosphoserine in >36,000 phosphosites by tandem mass spectrometry (Barber et al, Nat. Biotech. 2018). This phosphosite library was generated in a single mixed pool by expressing and purifying the phosphosite library as GST-fusion peptides (“mode #1” expression). The mode #1 phosphosite library (111704 Available Soon) is available from Addgene. This library is anticipated to be useful for laboratories performing phosphoproteomic experiments in need of reference spectra or phosphopeptide standards for quantitative assessments. The mode #1 phosphosite library can be generated with either phosphoserine using SepOTSλ (68292) or serine using supD tRNA (68307) at the central position.

Introducing Hi-P for screening phosphorylation-dependent protein-protein interactions. The phosphosite DNA library can also be introduced into a second expression vector encoding a split mCherry fluorescent reporter, enabling the detection of phosphorylation-dependent protein-peptide interactions (“mode #2” expression, Barber et al, Nat. Biotech. 2018). The phosphosite library is fused to the N-terminal portion of split mCherry, while a phosphobinding domain is fused to the C-terminal mCherry fragment. Upon interaction between the phosphobinding domain and the phosphosite, mCherry fluorescence is restored, so interactions can be discovered by fluorescence-activated cell sorting (FACS). The mode #2 phosphosite library with four different phosphobinding domains (14-3-3β, 14-3-3σ, the WW2 domain of NEDD4, and the WW2 domain of NEDD4-2; 111705-8 - Available Soon) is available from Addgene.

Diagram of mode #1 and mode #2 in the Rinehart Phosphoprotein library

Screening kinases. The mode #1 phosphosite library can also be synthesized using supD tRNA (68307) to generate the phosphosites with serine instead of phosphoserine (i.e. a “phosphorylatable” phosphosite library). This serine phosphosite library can then be used to identify candidate human substrates of kinases of interest by mass spectrometry in a technique called serine-oriented library/kinase-library reactions (SERIOHL-KILR, Barber et al, Biochemistry 2018).

Original Rinehart & Söll Kit (ca. 2011) requires: Improved Reagents (ca. 2015) requires:
E. coli strain: BL21ΔserB (34929) E. coli strain: C321.ΔA.ΔserB.Amp S (68306)
Vectors:
  • pKD-SepRS-EFSep (34623)
  • pCAT112TAG-SepT (34624)
  • Third vector with gene of interest (e.g.68295 or 53225)
Vectors:

Rinehart Lab Improved Phosphoprotein Synthesis Reagents

Type ID Item
Strain 68306 C321.ΔA.Δserb.Amp S
Plasmid 68292 SepOTSλ
Plasmid 68307 SupD
Plasmid 34623 SepOTSα SepRS/EF-Sep ( aka. pKD-SepRS-EFSep)
Plasmid 34624 SepOTSα tRNA-Sep ( aka. pCAT112TAG-SepT)
Plasmid 68283 SepOTSβ
Plasmid 68284 SepOTSγ
Plasmid 68285 SepOTSδ
Plasmid 68286 SepOTSϵ
Plasmid 68287 SepOTSζ
Plasmid 68288 SepOTSη
Plasmid 68289 SepOTSθ
Plasmid 68290 SepOTSι
Plasmid 68291 SepOTSκ
Plasmid 52054 SepOTSµ (aka. B40 OTS)
Plasmid 68294 SepOTSν
Plasmid 68295 GFP E17TAG
Plasmid 68296 GFP S2TAG
Plasmid 68297 GFP Q157TAG
Plasmid 68298 GFP S2TAG/E17TAG
Plasmid 68299 GFP E17TAG/Q157TAG
Plasmid 53225 MBP-MEK1 S218TAG/S222TAG ( aka. PCRT7 tetR pLtetO MBP-MEK1 XX Amp)
Plasmid 68300 MBP-MEK1
Plasmid 68301 MBP-MEK1 S218TAG
Plasmid 68302 MBP-MEK1 S222TAG
Plasmid 68305 Beta lactamase S68TAG
Plasmid 69118 E17TAG GFP zeo resistance
Strain 34929 BL21ΔserB
Strain 52055 EcAR7
Plasmid 112031 pNAS1b split mCherry 14-3-3β ctrl
Plasmid 112030 pNAS1b split mCherry NEDD4 WW2 neg ctrl
Plasmid 112029 pNAS1b split mCherry NEDD4 WW2 pos ctrl
Plasmid 111883 pNAS1b split mCherry NEDD4-2 WW2
Plasmid 111882 pNAS1b split mCherry NEDD4 WW2
Plasmid 111881 pNAS1b split mCherry 14-3-3σ
Plasmid 111880 pNAS1b split mCherry 14-3-3β
Pooled Library 111704 Mode #1 Library
Pooled Library 111705 Mode #2 Library (14-3-3β)
Pooled Library 111706 Mode #2 Library (14-3-3σ)
Pooled Library 111707 Mode #2 Library (NEDD4 WW2 domain)
Pooled Library 111708 Mode #2 Library (NEDD4-2 WW2 domain)

References & Protocols

Human Serine Phosphopeptide Library Described in:

Encoding human serine phosphopeptides in bacteria for proteome-wide identification of phosphorylation-dependent interactions. Barber KW, Muir P, Szeligowski RV, Rogulina S, Gerstein M, Sampson JR, Isaacs FJ, Rinehart J. Nat Biotechnol. 2018 Aug;36(7):638-644. PubMed

Kinase Substrate Profiling Using a Proteome-wide Serine-Oriented Human Peptide Library. Barber KW, Miller CJ, Jun JW, Lou HJ, Turk BE, Rinehart J. 2018. Biochemistry. 2018 Aug 7;57(31):4717-4725. PubMed

Improved Reagents (ca. 2015) Described in:

A flexible codon in genomically recoded Escherichia coli permits programmable protein phosphorylation. Pirman NL, Barber KW, Ma NJ, Haimovich AD, Rogulina S, Isaacs FJ, and Rinehart J. Nature Communications. 2015. 6, 8130. PubMed

Robust Production of Recombinant Phosphoproteins Using Cell-Free Protein Synthesis. Oza JP, Aerni HR, Pirman NL, Rogulina S, ter Haar CM, Isaacs FJ, Rinehart J, and Jewett MC. Nature Communications. 2015. 6, 8168. PubMed

The PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives a Program of OPTN/NDP52 Recruitment and TBK1 Activation to Promote Mitophagy. Heo JM, Ordureau A, Paulo JA, Rinehart J, Harper JW. Molecular Cell. 2015. Sep 9. pii: S1097-2765(15)00662-0. doi: 10.1016/j.molcel.2015.08.016. PubMed

Protocol for using the Improved Reagents (ca. 2015) from the Rinehart Lab:

Icon Users Guide for Improved 2015 Phosphoprotein Reagents (525.9 KB)

Original Kit (ca. 2011) Described in:

Expanding the genetic code of Escherichia coli with phosphoserine. Park HS, Hohn MJ, Umehara T, Guo LT, Osborne EM, Benner J, Noren CJ, Rinehart J, Söll D. Science. 2011 Aug 26;333(6046):1151-4. PubMed

Reagents (ca. 2014) Described in:

Enhanced phosphoserine insertion during Escherichia coli protein synthesis via partial UAG codon reassignment and release factor 1 deletion. Heinemann IU, Rovner AJ, Aerni HR, Rogulina S, Cheng L, Olds W, Fischer JT, Söll D, Isaacs FJ, Rinehart J. FEBS Lett. 2012. Oct 19;586(20):3716-22. PubMed

Protocol for Reagents (ca. 2014) from the Rinehart Lab:

Icon Users Guide for 2014 Phosphoprotein Reagents (691.3 KB)