The development of MassM2 single-chain variable fragment (ScFv) antibody specific to FLAG peptide
2022
Kim, J.K.
An anti-FLAG-M2 mouse monoclonal antibody specific to the FLAG peptide (DYKDDDDK) is widely used worldwide to purify or detect FLAG-tagged proteins. However, the amino acid sequences involved in antigen binding was unknown. Recently, the amino acid sequences of the variable domain, which is involved in the antigen binding, has been determined through X-ray crystallography and Mass spectroscopy. Therefore, in this study, the amino acid sequences of the heavy and light chain variable domains named as MassM2 were newly redesigned by aligning the amino acid sequences of the heavy and light chain variable domains determined by two methods mentioned above. In addition, the MassM2 three-dimensional structure predicted by computational modeling program called as AlphaFold and the already reported 2G60 crystal structure were superimposed to calculate RMSD (Root Mean Square Deviation) for comparison of structural similarity. Depending on the newly determined variable heavy and light chain amino acid sequences of the MassM2 antibody, corresponding genes were designed, synthesized, and cloned into the pUC119 expression vector and then expressed in Escherichia coli as a recombinant MassM2 single chain variable fragment (ScFv) antibody. This antibody showed binding activity to both FLAG peptide and FLAG peptide tagged protein in enzyme-linked immunosorbent assay and Western blot. In addition, the dissociation equilibrium constant (KD) were calculated as 1.12 × 10⁻⁸ M–1.342 × 10⁻⁷ M through bio-layer interferometry confirming antigen binding activity.
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