Traceless and site-specific ubiquitination of recombinant proteins

Satpal Virdee; Prashant B. Kapadnis; Thomas Elliott; Kathrin Lang; Julia Madrzak; Duy P. Nguyen; Lutz Riechmann; Jason W. Chin

doi:10.1021/ja202799r

Traceless and site-specific ubiquitination of recombinant proteins

Satpal Virdee, Prashant B. Kapadnis, Thomas Elliott, Kathrin Lang, Julia Madrzak, Duy P. Nguyen, Lutz Riechmann, Jason W. Chin (Lead / Corresponding author)

Research output: Contribution to journal › Article › peer-review

159 Citations (Scopus)

Abstract

Protein ubiquitination is a post-translational modification that regulates almost all aspects of eukaryotic biology. Here we discover the first routes for the efficient site-specific incorporation of delta-thiol-L-lysine (7) and delta-hydroxy-L-lysine (8) into recombinant proteins, via evolution of a pyrrolysyl-tRNA synthetase/tRNA(CUA) pair. We combine the genetically directed incorporation of 7 with native chemical ligation and desulfurization to yield an entirely native isopeptide bond between substrate proteins and ubiquitin. We exemplify this approach by demonstrating the synthesis of a ubiquitin dimer and the first synthesis of ubiquitinated SUMO.

Original language	English
Pages (from-to)	10708-10711
Number of pages	4
Journal	Journal of the American Chemical Society
Volume	133
Issue number	28
DOIs	https://doi.org/10.1021/ja202799r
Publication status	Published - 20 Jul 2011

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abstract = "Protein ubiquitination is a post-translational modification that regulates almost all aspects of eukaryotic biology. Here we discover the first routes for the efficient site-specific incorporation of delta-thiol-L-lysine (7) and delta-hydroxy-L-lysine (8) into recombinant proteins, via evolution of a pyrrolysyl-tRNA synthetase/tRNA(CUA) pair. We combine the genetically directed incorporation of 7 with native chemical ligation and desulfurization to yield an entirely native isopeptide bond between substrate proteins and ubiquitin. We exemplify this approach by demonstrating the synthesis of a ubiquitin dimer and the first synthesis of ubiquitinated SUMO.",

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AU - Virdee, Satpal

AU - Kapadnis, Prashant B.

AU - Elliott, Thomas

AU - Lang, Kathrin

AU - Madrzak, Julia

AU - Nguyen, Duy P.

AU - Riechmann, Lutz

AU - Chin, Jason W.

PY - 2011/7/20

Y1 - 2011/7/20

N2 - Protein ubiquitination is a post-translational modification that regulates almost all aspects of eukaryotic biology. Here we discover the first routes for the efficient site-specific incorporation of delta-thiol-L-lysine (7) and delta-hydroxy-L-lysine (8) into recombinant proteins, via evolution of a pyrrolysyl-tRNA synthetase/tRNA(CUA) pair. We combine the genetically directed incorporation of 7 with native chemical ligation and desulfurization to yield an entirely native isopeptide bond between substrate proteins and ubiquitin. We exemplify this approach by demonstrating the synthesis of a ubiquitin dimer and the first synthesis of ubiquitinated SUMO.

AB - Protein ubiquitination is a post-translational modification that regulates almost all aspects of eukaryotic biology. Here we discover the first routes for the efficient site-specific incorporation of delta-thiol-L-lysine (7) and delta-hydroxy-L-lysine (8) into recombinant proteins, via evolution of a pyrrolysyl-tRNA synthetase/tRNA(CUA) pair. We combine the genetically directed incorporation of 7 with native chemical ligation and desulfurization to yield an entirely native isopeptide bond between substrate proteins and ubiquitin. We exemplify this approach by demonstrating the synthesis of a ubiquitin dimer and the first synthesis of ubiquitinated SUMO.

U2 - 10.1021/ja202799r

DO - 10.1021/ja202799r

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SP - 10708

EP - 10711

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

IS - 28

ER -

Traceless and site-specific ubiquitination of recombinant proteins

Abstract

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