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<p>Protein–protein interaction analysis requires not only the detection of a single protein but also multiple interacting proteins. The proximity-dependent biotin identification (BioID) method using biotin proximity-labelled enzymes is a protein-protein interaction method used in cells and in vivo; however, the detection tags are limited because even the lysine residues of the amino acids in the detection tag are labelled by the enzyme. Herein, we demonstrated a new affinity tag system without lysine residues suitable for use in the BioID method. After immunisation of rabbits with <i>Plasmodium falciparum</i> PfRipr5 protein, 22 anti-PfRipr5 monoclonal antibodies were isolated by ImmunoSpot Array Assay on a Chip, and their binding and specificity were analysed. The results showed that No. 6 anti-PfRipr5 monoclonal antibody had the highest sensitivity and specificity. The epitope was identified to contain one lysine residue; therefore, the mutated epitope, which was replaced with an arginine residue, showed similar binding properties. It was named REC tag for further analysis, and the REC tag was compatible with IB, AlphaScreen, and immunostaining and showed resistance to biotinylation using TurboID in vitro. These results indicate that a new detection tag system could be developed, in which REC tags could be used to detect BioID methods.</p>

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A lysine-free REC tag system for proximity-biotinylation applications

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月8日

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  • Soh Tokunaga,
  • Hikaru Nagaoka,
  • Tatsuhiko Ozawa,
  • Kohdai Yamada,
  • Hiroyuki Takeda,
  • Hinako Fujisaki,
  • Mana Hamada,
  • Fumiya Soga,
  • Hiroyuki Kishi,
  • Eizo Takashima,
  • Tatsuya Sawasaki

摘要

Protein–protein interaction analysis requires not only the detection of a single protein but also multiple interacting proteins. The proximity-dependent biotin identification (BioID) method using biotin proximity-labelled enzymes is a protein-protein interaction method used in cells and in vivo; however, the detection tags are limited because even the lysine residues of the amino acids in the detection tag are labelled by the enzyme. Herein, we demonstrated a new affinity tag system without lysine residues suitable for use in the BioID method. After immunisation of rabbits with Plasmodium falciparum PfRipr5 protein, 22 anti-PfRipr5 monoclonal antibodies were isolated by ImmunoSpot Array Assay on a Chip, and their binding and specificity were analysed. The results showed that No. 6 anti-PfRipr5 monoclonal antibody had the highest sensitivity and specificity. The epitope was identified to contain one lysine residue; therefore, the mutated epitope, which was replaced with an arginine residue, showed similar binding properties. It was named REC tag for further analysis, and the REC tag was compatible with IB, AlphaScreen, and immunostaining and showed resistance to biotinylation using TurboID in vitro. These results indicate that a new detection tag system could be developed, in which REC tags could be used to detect BioID methods.