title: Segmental isotopic labeling of a single-domain globular protein without any refolding step by an asparaginyl endopeptidase.
abstract: Asparaginyl endopeptidases (AEPs) catalyze head-to-tail backbone cyclization of naturally occurring cyclic peptides such as cyclotides, and have become an important peptide-engineering tool for macrocyclization and peptide ligation. Here, we report efficient protein ligation in trans by mimicking efficient backbone cyclization by an AEP without any excess of reactants. We demonstrate a practical application of segmental isotopic labeling for NMR studies of a single-domain globular protein without any refolding step using the recombinant AEP prepared from Escherichia coli. This simple protein ligation approach using an AEP could be applied for incorporation of various biophysical probes into proteins as well as post-translational production of full-length proteins.
authors.affiliation: Research Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Finland.
authors.forename: Kornelia M
authors.initials: KM
authors.lastname: Mikula
authors.affiliation: Research Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Finland.
authors.forename: Igor
authors.initials: I
authors.lastname: Tascón
authors.affiliation: Research Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Finland.
authors.forename: Jenni J
authors.initials: JJ
authors.lastname: Tommila
authors.affiliation: Research Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, Finland.
authors.forename: Hideo
authors.initials: H
authors.lastname: Iwaï
mesh_terms: D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D007553:Isotope Labeling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D032067:Oldenlandia / Q000201:enzymology / Q000235:genetics; D010456:Peptides, Cyclic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D000099240:Asparaginyl Endopeptidase
chemical_list: D003546:Cysteine Endopeptidases; D010456:Peptides, Cyclic; D010940:Plant Proteins; D011994:Recombinant Proteins; D000099240:Asparaginyl Endopeptidase
keywords: 
  NMR
  ; asparaginyl endopeptidase; protein ligation; segmental isotopic labeling