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Segmental isotopic labeling by asparaginyl endopeptidase-mediated protein ligation

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Abstract

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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Abbreviations

PTS:

Protein-trans splicing

CTS:

Conditional protein trans-splicing

EPL:

Expressed protein ligation

IPL:

Intein-mediated protein ligation

NCL:

Native chemical ligation

SML:

Sortase-mediated ligation

AEP:

Asparaginyl endopeptidase

AML:

AEP-mediated ligation

SrtA:

Staphylococcus aureus sortase A

HSQC:

Heteronuclear single-quantum correlation spectroscopy

OaAEP1:

Oldenlandia affinis asparaginyl endopeptidase 1b

HINT domain:

Hedgehog/intein domain

dArmRP:

Designed armadillo repeat protein

GFP:

Green fluorescent protein

MBP:

Maltose-binding protein

Ulp1:

Ubiquitin-like-specific protease 1

IMAC:

Immobilized metal ion affinity chromatography

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

CBD:

Chitin-binding domain

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Acknowledgements

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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Authors and Affiliations

  1. Research Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, P.O. Box 65, 00014, Helsinki, Finland

    Kornelia M. Mikula, Luisa Krumwiede & Hideo Iwaï

  2. Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, 8057, Zurich, Switzerland

    Andreas Plückthun

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  1. Kornelia M. Mikula
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  2. Luisa Krumwiede
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  3. Andreas Plückthun
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  4. Hideo Iwaï
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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). https://doi.org/10.1007/s10858-018-0175-4

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