A Facile Strategy for Selective Incorporation of Phosphoserine into Histones

Lee, Sangsik; Oh, Shi-Hwan; Yang, Aerin; Kim, Jihyo; Söll, Dieter; Lee, Daeyoup; Park, Hee-Sung

doi:10.1002/anie.201300531

Cited by 93 publications

(95 citation statements)

References 21 publications

(26 reference statements)

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“…To enhance production of different pUb proteins, we introduced 2nd generation pSer system mutations [26] into the pKD-pSer1 plasmid to yield pDS-pSer2. This pSer insertion system is more efficient and able to compete against RF1 [26], so the protein production host was switched to BL21(DE3).…”

Section: Characterization Of Purified Phosphorylated Ubmentioning

confidence: 99%

“…This pSer insertion system is more efficient and able to compete against RF1 [26], so the protein production host was switched to BL21(DE3). Because nearcognate decoding of UAG is minimal in BL21(DE3) (Fig.…”

Section: Characterization Of Purified Phosphorylated Ubmentioning

confidence: 99%

“…Previous reports have shown it is possible to produce designer phosphoprotein in E. coli [13,26], but the efficient production of pure phosphoprotein has remained challenging. We successfully produced several pUb variants in unambiguously pure form as evidenced by MS/MS and Phos-tag gels.…”

Section: Pure Designer Phosphoproteinmentioning

confidence: 99%

“…Our goal in this study was not to necessarily maximize the yield of phosphoprotein. We [17] (Table S1) and others [11,22,26] have already produced phosphoproteins in sufficient amounts for biochemical and structural studies. Rather, we were concerned with demonstrating unambiguously and in a more rigorous fashion than previous reports that we indeed obtained pure phosphoprotein.…”

Section: Pure Designer Phosphoproteinmentioning

confidence: 99%

“…Previous work relied on MS/MS identification of pSer in the phosphoprotein [11,26], yet mistranslated protein may go undetected with this method. A modified pSer system [11], including mutations in SepRS and the anticodon stem of tRNA Sep , recently reported similar mgÁL À1 culture yields of phosphoprotein as was previously reported for the 2nd generation pSer system [26]. While our yields were somewhat lower, we emphasize that we are reporting pure phosphoprotein yields, which likely reflect some loss of protein during column chromatography.…”

Section: Pure Designer Phosphoproteinmentioning

confidence: 99%

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Generation of phospho‐ubiquitin variants by orthogonal translation reveals codon skipping

et al. 2016

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Edited by Michael IbbaThe activity of the Parkinson's disease-linked E3 ligase parkin is stimulated by phosphorylation at ubiquitin Ser65 (pUb S65 ). The role of other ubiquitin phospho-sites and their kinases are unknown. We produced pUb variants (pS7, pS12, pS20, pS57, pS65) by genetically encoding phosphoserine with the UAG codon. In release factor-deficient Escherichia coli (DRF1), intended to enhance UAG read-through, we discovered ubiquitin variants lacking the UAG-encoded residue, demonstrating previously undocumented +3 frame shifting. We successfully purified each pUb variant from mistranslated products. While pUb S20 failed to stimulate parkin, parkin was partially active with pUb S12 . We observed significant ubiquitination when pUb S65 was the sole substrate.

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Section: Characterization Of Purified Phosphorylated Ubmentioning

confidence: 99%

Section: Characterization Of Purified Phosphorylated Ubmentioning

confidence: 99%

Section: Pure Designer Phosphoproteinmentioning

confidence: 99%

Section: Pure Designer Phosphoproteinmentioning

confidence: 99%

Section: Pure Designer Phosphoproteinmentioning

confidence: 99%

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Generation of phospho‐ubiquitin variants by orthogonal translation reveals codon skipping

et al. 2016

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Expanding the genetic code of Escherichia coli with phosphotyrosine

Fan

Söll

2016

FEBS Letters

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Protein phosphorylation is one of the most important post-translational modifications in nature. However, the site-specific incorporation of O-phosphotyrosine into proteins in vivo has not yet been reported. Endogenous phosphatases present in cells can dephosphorylate phosphotyrosine as a free amino acid or as a protein residue. Therefore, we deleted the genes of five phosphatases from the genome of Escherichia coli with the aim of stabilizing phosphotyrosine. Together with an engineered aminoacyl-tRNA synthetase (derived from Methanocaldococcus jannaschii tyrosyl-tRNA synthetase) and an elongation factor Tu variant, we were able to co-translationally incorporate O-phosphotyrosine into the super-folder green fluorescent protein at a desired position in vivo. This system will facilitate future studies of tyrosine phosphorylation.

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Selenocysteine

Rother

2015

Encyclopedia of Life Sciences

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The trace element selenium is present in the form of selenocysteine in selected proteins of organisms from all three domains of life. Most of the selenoproteins (i.e. proteins containing selenocysteine) investigated thus far are oxidoreductases with selenocysteine present in the active site and playing a crucial role in – or creating the required environment for – catalysis. Selenocysteine incorporation is DNA encoded, determined by the UGA codon, and occurs via a unique mechanism representing an expansion of the genetic code. Despite the conceptual conservation of the principal mechanism of selenocysteine synthesis and incorporation in all lines of descent, the bacterial system differs from the archaeal/eukaryal system. Detailed biochemical and structural knowledge of selenoprotein synthesis has allowed rewiring bacterial translation for targeted selenocysteine insertion. Key Concepts Selenium is an essential trace element for many organisms including humans. The most important form of biologically active selenium is the amino acid selenocysteine. Selenocysteine is co‐translationally inserted into growing polypeptides. Selenocysteine insertion proceeds via a unique mechanism recoding UGA. The mechanism of selenocysteine insertion differs in Bacteria, Archaea, and Eukarya.

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A Facile Strategy for Selective Incorporation of Phosphoserine into Histones

Cited by 93 publications

References 21 publications

Generation of phospho‐ubiquitin variants by orthogonal translation reveals codon skipping

Generation of phospho‐ubiquitin variants by orthogonal translation reveals codon skipping

Expanding the genetic code of Escherichia coli with phosphotyrosine

Selenocysteine

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