Conformational and functional properties of an undecapeptide epitope fused with the C-terminal end of the maltose binding protein

Monoclonal antibody mAb164 is directed against the TrpB₂ subunit of the Escherichia coli tryptophan synthase. It recognizes the synthetic peptide P11, constituted of residues 273-283 of TrpB, with high affinity. We constructed a hybrid protein in which the C-terminal end of protein Ma1E was linked with the N-terminal end of P11. Hybrid Ma1E-P11 was produced in E. coli from a plasmidic gene and purified in one step as Ma1E. Ma1E-P11 and the isolated P11 had identical conformational and functional properties according to the following criteria. The NMR spectra of Ma1E and Ma1E-P11 in TOCSY experiments showed that the P11 moiety of Ma1E-P11 moved independently from its Ma1E moiety. The chemical shifts of the protons for the P11 moiety of Ma1E-P11 and for the isolated P11 were very close and did not show significant deviations from random coil values. The equilibrium constant of dissociation (K(D)) from mAb164, measured by a competition ELISA, was identical for Ma1E-P11 and the isolated P11, around 6 nM. The change of the C-terminal residue of Ma1E-P11 from Lys into Ala increased 37-fold this dissociation constant. This increase showed that the P11 moiety of Ma1E-P11 was not degraded. The high molecular mass of Ma1E-P11 allowed us to follow its kinetics of interaction with immobilized mAb164 by surface plasmon resonance, using the BIAcore apparatus. The rates of association with mAb164 were similar for Ma1E-P11 and TrpB₂, but the dissociation was faster for Ma1E-P11 than for TrpB₂, as previously observed for the isolated P11 by a fluorometric method. Thus, the fusion of peptides with the C-terminal end of Ma1E could constitute an alternative to chemical synthesis for the study of their recognition by receptors, in vivo or in vitro.