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Segmental isotopic labeling by asparaginyl endopeptidase-mediated protein ligation


Segmental isotopic labeling can facilitate NMR studies of large proteins, multi-domain proteins, and proteins with repetitive sequences by alleviating NMR signal overlaps. Segmental isotopic labeling also allows us to investigate an individual domain in the context of a full-length protein by NMR. Several established methods are available for segmental isotopic labeling such as intein-mediated ligation, but each has specific requirements and limitations. Here, we report an enzymatic approach using bacterially produced asparagine endopeptidase from Oldenlandia affinis for segmental isotopic labeling of a protein with repetitive sequences, a designed armadillo repeat protein, by overcoming some of the shortcomings of enzymatic ligation for segmental isotopic labeling.

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Protein-trans splicing


Conditional protein trans-splicing


Expressed protein ligation


Intein-mediated protein ligation


Native chemical ligation


Sortase-mediated ligation


Asparaginyl endopeptidase


AEP-mediated ligation


Staphylococcus aureus sortase A


Heteronuclear single-quantum correlation spectroscopy


Oldenlandia affinis asparaginyl endopeptidase 1b

HINT domain:

Hedgehog/intein domain


Designed armadillo repeat protein


Green fluorescent protein


Maltose-binding protein


Ubiquitin-like-specific protease 1


Immobilized metal ion affinity chromatography


Isopropyl β-d-1-thiogalactopyranoside


Chitin-binding domain


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This work is supported by grants from the Academy of Finland (137995, 277335). The NMR facility at the Institute of Biotechnology, University of Helsinki is supported by Biocenter Finland and Helsinki Institute of Life Science (HiLIFE). The authors thank K. Elsner, A. Hietikko, S. Jäskeläinen, J. Tommila, and E. Maschke for their technical assistance.

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Correspondence to Hideo Iwaï.

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Mikula, K.M., Krumwiede, L., Plückthun, A. et al. Segmental isotopic labeling by asparaginyl endopeptidase-mediated protein ligation. J Biomol NMR 71, 225–235 (2018).

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  • Asparaginyl endopeptidase
  • Designed armadillo repeat protein
  • Segmental isotopic labeling
  • Protein trans-ligation
  • Protein NMR