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Rinehart-icon.jpg Rinehart Lab reagents for improved expression of recombinant phosphoproteins


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 (S P 218, S P 222).

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).

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.68295or53225)
Vectors:

Rinehart Lab Improved Phosphoprotein Synthesis Reagents

References & Protocols

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)