Site-Specific Protein Labeling and Generation of Defined Ubiquitin-Protein Conjugates Using an Asparaginyl Endopeptidase
Abstract
Asparaginyl endopeptidases (AEPs) have recently been widely utilized for peptide and protein modification. Labeling is however restricted to protein termini, severely limiting flexibility and scope in creating diverse conjugates as needed for therapeutic and diagnostic applications. Here, we use genetic code expansion to site-specifically modify target proteins with an isopeptide-linked glycylglycine moiety that serves as an acceptor nucleophile in AEPmediated transpeptidation with various probes containing a tripeptidic recognition motif. Our approach allows simple and flexible labeling of recombinant proteins at any internal site and leaves a minimal, entirely peptidic footprint (NGG) in the conjugation product. We show site-specific labeling of diverse target proteins with various biophysical probes, including dual labeling at an internal site and the N-terminus. Furthermore, we harness AEP-mediated transpeptidation for generation of ubiquitinand ubiquitin-like-modifier conjugates bearing a native isopeptide bond and only one point mutation in the linker region. Show more
Permanent link
https://doi.org/10.3929/ethz-b-000561774Publication status
publishedExternal links
Journal / series
Journal of the American Chemical SocietyVolume
Pages / Article No.
Publisher
American Chemical SocietyOrganisational unit
09740 - Lang, Kathrin / Lang, Kathrin
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