title: Conformational and functional properties of an undecapeptide epitope fused with the C-terminal end of the maltose binding protein.
abstract: 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 MalE was linked with the N-terminal end of P11. Hybrid MalE-P11 was produced in E. coli from a plasmidic gene and purified in one step as MalE. MalE-P11 and the isolated P11 had identical conformational and functional properties according to the following criteria. The NMR spectra of MalE and MalE-P11 in TOCSY experiments showed that the P11 moiety of MalE-P11 moved independently from its MalE moiety. The chemical shifts of the protons for the P11 moiety of MalE-P11 and for the isolated P11 were very close and did not show significant deviations from random coil values. The equilibrium constant of dissociation (KD) from mAb164, measured by a competition ELISA, was identical for MalE-P11 and the isolated P11, around 6 nM. The change of the C-terminal residue of MalE-P11 from Lys into Ala increased 37-fold this dissociation constant. 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 resonance, using the BIAcore apparatus. The rates of association with mAb164 were similar for MalE-P11 and TrpB2, but the dissociation was faster for MalE-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 MalE could constitute an alternative to chemical synthesis for the study of their recognition by receptors, in vivo or in vitro.
authors.affiliation: Unité de Biochimie Cellulaire and Laboratoire de Résonance Magnétique Nucléaire, CNRS URA 1129, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
authors.forename: P
authors.initials: P
authors.lastname: Rondard
authors.forename: F
authors.initials: F
authors.lastname: Brégégère
authors.forename: A
authors.initials: A
authors.lastname: Lecroisey
authors.forename: M
authors.initials: M
authors.lastname: Delepierre
authors.forename: H
authors.initials: H
authors.lastname: Bedouelle
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000818:Animals; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015374:Biosensing Techniques; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000378:metabolism*; D029968:Escherichia coli Proteins; D009682:Magnetic Resonance Spectroscopy; D008320:Maltose / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011487:Protein Conformation; D014367:Tryptophan Synthase / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D002352:Carrier Proteins; D000939:Epitopes; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C461599:maltose transport system, E coli; D008320:Maltose; D014367:Tryptophan Synthase