RT Journal Article
SR Electronic
T1 Structural basis for recognition of the FLAG-tag by anti-FLAG M2
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2024.03.25.586599
DO 10.1101/2024.03.25.586599
A1 Beugelink, J. Wouter
A1 Sweep, Els
A1 Janssen, Bert J.C.
A1 Snijder, Joost
A1 Pronker, Matti F.
YR 2024
UL http://biorxiv.org/content/early/2024/03/28/2024.03.25.586599.abstract
AB The FLAG-tag/anti-FLAG system is a widely used biochemical tool for protein detection and purification. Anti-FLAG M2 is the most popular antibody against the FLAG-tag, due to its ease of use, versatility, and availability in pure form or as bead conjugate. M2 binds N-terminal, C-terminal and internal FLAG-tags and binding is calcium-independent, but the molecular basis for the FLAG-tag specificity and recognition remains unresolved.Here we present an atomic resolution (1.17 Å) structure of the FLAG peptide in complex with the Fab of anti-FLAG M2, revealing key binding determinants. Five of the eight FLAG peptide residues form direct interactions with paratope residues. The FLAG peptide adopts a 310 helix conformation in complex with the Fab. These structural insights allowed us to rationally introduce point mutations on both the peptide and antibody side. We tested these by surface plasmon resonance, leading us to propose a shorter yet equally binding version of the FLAG-tag for the M2 antibody.Competing Interest StatementThe authors have declared no competing interest.