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

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Protein-protein interaction (PPI) analysis is crucial for understanding cellular processes.
• Proximity-dependent biotin identification (BioID) is a key method for mapping PPIs.
• Existing BioID tags have limitations due to lysine residues being susceptible to biotinylation, hindering accurate detection.

Purpose of the Study:

• To develop a novel, lysine-free affinity tag for enhanced BioID applications.
• To create a tag resistant to self-biotinylation for improved PPI mapping.
• To validate the compatibility and performance of the new tag in various biological assays.

Main Methods:

• Development of a lysine-free affinity tag (REC tag) derived from anti-Plasmodium falciparum PfRipr5 monoclonal antibodies.
• Mutation of a lysine residue in the epitope to arginine to create the REC tag.
• Testing the REC tag's compatibility with immunoblotting (IB), AlphaScreen, and immunostaining.
• Evaluating the REC tag's resistance to biotinylation using TurboID in vitro.

Main Results:

• A highly sensitive and specific anti-PfRipr5 monoclonal antibody (No. 6) was identified.
• A mutated epitope (REC tag) retained binding properties without lysine residues.
• The REC tag demonstrated compatibility with IB, AlphaScreen, and immunostaining.
• The REC tag exhibited resistance to in vitro biotinylation by TurboID.

Conclusions:

• The REC tag represents a significant advancement for BioID methods, overcoming lysine-related limitations.
• This new tag enables more precise and reliable protein-protein interaction studies.
• The REC tag has broad applicability in various biochemical and cellular analyses, including in vivo studies.