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Rinehart Lab Phosphoprotein Reagents

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). Follow up work from the Rinehart and Issacs labs at Yale have made improvements to this system by genomically recoding E.coli (Lajoie et al., Science 2013) and improving the orthogonal translation system to make singly or multiply phosphorylated proteins (Pirman et al., Nat Comm 2015).

Improved phosphoprotein production with rEcoli XpS and a new pSerOTS. Recently the Rinehart lab published a new strain of recoded E.coli (rEcoli XpS 192872) and an optimized phosphoserine orthogonal translation system (pSerOTS-C1* (V70) 188537). These reagents are described in detail in (Mohler et al., Mol Syst Biol 2023) and help deliver improved phosphoprotein yield, rEcoli growth characteristics, and an overall improved ease of use when expressing recombinant phosphoproteins. These attributes make rEcoli XpS (192872) and pSerOTS-C1* (188537) a better choice in place of the popular SepOTSλ (68292). An important technical note: pSerOTS-C1* (188537) is a ROP minus plasmid and is not compatible with other plasmids containing high copy ColE1/PUC-like origins. Therefore, when choosing plasmids to express the phosphoprotein of interest, a medium copy RSF1030 origins or a low copy p15a origins should be used. Find further details in Mohler et al., Mol Syst Biol 2023 and find general tips on phosphoprotein expression in rEcoli XpS in the Methods section link below (Methods for iSPI, rEcoli XPS, pSerOTS-C1*).

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). 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) 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) 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).

Iterative Synthetically Phosphorylated Isomers (iSPI). This new resource described in (Gassaway et al., Nature Methods 2022) is adapted from the synthetic human phosphoproteome. The iSPI resource subdivides the 110,139 human phosphoserine site library (188536) into 10 subpools of ~11,000 phosphosites (188526; 188527; 188528; 188529; 188530; 188531; 188532; 188533; 188534; 188535). Importantly, phosphorylation positional isomers are allocated to different subpools, allowing precise knowledge of phosphorylated positions for testing phosphoproteomics methods, proteomics search algorithms, proteomics phosphosite localization algorithms, or any method confounded by phosphosite ambiguity.

Rinehart Lab Improved Phosphoprotein Synthesis Reagents

Type ID Item
Strain 192872 rEcoli XpS (MG1655-C321 mutS+, λ-, Δ(ybhB-bioAB)::zeoR, ΔprfA, ΔserB)
Plasmid 188537 pSerOTS-C1* (V70)
Pooled Library 188526 iSPI_pSer_Subpool#1
Pooled Library 188527 iSPI_pSer_Subpool#2
Pooled Library 188528 iSPI_pSer_Subpool#3
Pooled Library 188529 iSPI_pSer_Subpool#4
Pooled Library 188530 iSPI_pSer_Subpool#5
Pooled Library 188531 iSPI_pSer_Subpool#6
Pooled Library 188532 iSPI_pSer_Subpool#7
Pooled Library 188533 iSPI_pSer_Subpool#8
Pooled Library 188534 iSPI_pSer_Subpool#9
Pooled Library 188535 iSPI_pSer_Subpool#10
Pooled Library 188536 iSPI_pSer_Full_library
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

Iterative Synthetically Phosphorylated Isomers (iSPI) Described in:

A multi-purpose, regenerable, proteome-scale, human phosphoserine resource for phosphoproteomics. Gassaway BM, Li J, Rad R, Mintseris J, Mohler K, Levy T, Aguiar M, Beausoleil SA, Paulo JA, Rinehart J, Huttlin EL, Gygi SP. Nat Methods 2022 Nov;19(11):1371-1375. doi: 10.1038/s41592-022-01638-5. PubMed

rEcoli XpS and a new pSerOTS Described in:

System-wide optimization of an orthogonal translation system with enhanced biological tolerance. Mohler K, Moen JM, Rogulina S, Rinehart J. Mol Syst Biol 2023 Aug 8;19(8):e10591. doi: 10.15252/msb.202110591. PubMed

Protocols for using iSPI, rEcoli XPS, and pSerOTS-C1* (V70) (ca. 2022) Described in:

Methods for iSPI, rEcoli XPS, pSerOTS-C1* (V70) 551 KB

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:

Users Guide for Improved 2015 Phosphoprotein Reagents 525.9 KB

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:

Users Guide for 2014 Phosphoprotein Reagents 691.3 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