ChemRxiv
These are preliminary reports that have not been peer-reviewed. They should not be regarded as conclusive, guide clinical practice/health-related behavior, or be reported in news media as established information. For more information, please see our FAQs.
1/1
0/0

Lysine Acylation Using Conjugating Enzymes (LACE) for Site-Specific Modification and Ubiquitination of Native Proteins

preprint
submitted on 23.10.2019 and posted on 25.10.2019 by Raphael Hofmann, Gaku Akimoto, Thomas G. Wucherpfennig, Cathleen Zeymer, Jeffrey Bode

Enzymes are powerful tools for post-translational protein labeling due to their high sequence specificity and mild reaction conditions. Many existing protocols, however, are restricted to conjugations at terminal positions or rely on non-peptidic metabolites and large recognition domains. Here we introduce a chemoenzymatic method to functionalize proteins at internal lysine residues that are part of genetically encoded minimal recognition tags (four residues). We achieved this by employing the intrinsic sequence specificity of the E2 SUMO-conjugating enzyme Ubc9 and a short peptide thioester, which together obviate the need for E1 and E3 enzymes. Using a range of protein substrates, we apply this approach to the conjugation of biochemical probes, one-pot dual-labeling reactions in combination with sortase, and site-specific monoubiquitination and ISG15ylation. The small tag size and large substrate tolerance of Ubc9 will make this a method of choice for protein engineering by isopeptide formation and the preparation of ubiquitinated proteins.

Funding

ETH Zürich

Stipendienfonds der Schweizerischen Chemischen Industrie (SSCI)

Nakajima Foundation Scholarship

History

Email Address of Submitting Author

bode@org.chem.ethz.ch

Institution

ETH Zürich

Country

Switzerland

ORCID For Submitting Author

0000-0001-8394-8910

Declaration of Conflict of Interest

J.W.B., R.H. and T.G.W. are co-inventors on a European patent application (19170647.2 - 1118) which incorporates discoveries described in this manuscript.

Exports