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 TrpB(2) subunit of the Escherichia coli tryptophan synthase. It recognizes the synthetic peptide P11, constituted of residues 273-283 of TrpB, with high affini ty. We constructed a hybrid protein in which the C-terminal end of pro tein MalE was linked with the N-terminal end of P11. Hybrid MalE-P11 w as produced in E. coli from a plasmidic gene and purified in one step as MalE. MalE-P11 and the isolated P11 had identical conformational an d functional properties according to the following criteria, The NMR s pectra of MalE and MalE-P11 in TOCSY experiments showed that the P11 m oiety of MalE-P11 moved independently from its MalE moiety. The chemic al shifts of the protons for the PII moiety of MalE-P11 and for the is olated P11 were very close and did not show significant deviations fro m random coil values. The equilibrium constant of dissociation (KD) fr om mAb164, measured by a competition ELISA, was identical for MalE-P11 and the isolated P11, around 6 nM. The change of the C-terminal resid ue of MalE-P11 from Lys into Ala increased 37-fold this dissociation c onstant. This increase showed that the P11 moiety of MalE-P11 was not degraded. The high molecular mass of MalE-P11 allowed us to follow its kinetics of interaction with immobilized mAb164 by surface plasmon re sonance, using the BIAcore apparatus. The rates of association with mA b164, were similar for MalE-P11 and TrpB(2), but the dissociation was faster for MalE-P11 than for TrpB(2), as previously observed for the i solated P11 by a fluorometric method. Thus, the fusion of peptides wit h the C-terminal end of MalE could constitute an alternative to chemic al synthesis for the study of their recognition by receptors, in vivo or in vitro.