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 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.
Rondard
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P
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.
Brégégère
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F
Lecroisey
A
A
Delepierre
M
M
Bedouelle
H
H
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*
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