Site-specific epitope insertion into recombinant proteins using the MAP tag system

Ayami Wakasa, Mika K. Kaneko, Yukinari Kato, Junichi Takagi, Takao Arimori

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

Abstract

The MAP tag system comprises a 14-residue peptide derived from mouse podoplanin and its high-affinity monoclonal antibody PMab-1. We determined the crystal structure of PMab-1 complexed with the MAP tag peptide and found that the recognition required only the N-terminal 8 residues of MAP tag sequence, enabling the shortening of the tag length without losing the affinity for PMab-1. Furthermore, the structure illustrated that the MAP tag adopts a U-shaped conformation when bound by PMab-1, suggesting that loop-inserted MAP tag would assume conformation compatible with the PMab-1 binding. We inserted the 8-residue MAP tag into multiple loop regions in various proteins including fibronectin type III domain and G-protein-coupled receptors and tested if they maintain PMab-1 reactivity. Despite the conformational restraints forced by the insertion position, all MAP-inserted mutants were expressed well in mammalian cells at levels comparable to the non-tagged proteins. Furthermore, the binding by PMab-1 was fully maintained even for the mutant where MAP tag was inserted at a structurally restricted b-hairpin, indicating that the MAP tag system has unique feature that allows placement in the middle of protein domain at desired locations. Our results indicate the versatile utility of the MAP tag system in ‘site-specific epitope insertion’ application.

Original languageEnglish
Pages (from-to)375-384
Number of pages10
JournalJournal of biochemistry
Volume168
Issue number4
DOIs
Publication statusPublished - 2020 Oct 1

Keywords

  • Antibody
  • Epitope tag system
  • Flow cytometry
  • G-protein-coupled receptor
  • X-ray crystallography

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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