Rondard P, Brégégère F, Lecroisey A, Delepierre M, Bedouelle H
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.
Biochemistry. 1997 Jul 22;36(29):8954-61. doi: 10.1021/bi962508d.
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.
单克隆抗体mAb164针对大肠杆菌色氨酸合酶的TrpB2亚基。它以高亲和力识别由TrpB的273 - 283位残基构成的合成肽P11。我们构建了一种杂合蛋白，其中MalE蛋白的C末端与P11的N末端相连。杂合MalE - P11由质粒基因在大肠杆菌中产生，并作为MalE一步纯化。根据以下标准，MalE - P11和分离出的P11具有相同的构象和功能特性。在TOCSY实验中，MalE和MalE - P11的核磁共振谱表明，MalE - P11的P11部分与其MalE部分独立移动。MalE - P11的P11部分和分离出的P11的质子化学位移非常接近，且与无规卷曲值无显著偏差。通过竞争ELISA测量，MalE - P11和分离出的P11与mAb164的解离平衡常数（KD）相同，约为6 nM。MalE - P11的C末端残基从赖氨酸变为丙氨酸使该解离常数增加了37倍。这种增加表明MalE - P11的P11部分未被降解。MalE - P11的高分子量使我们能够使用BIAcore仪器通过表面等离子体共振跟踪其与固定化mAb164的相互作用动力学。MalE - P11和TrpB2与mAb164的结合速率相似，但MalE - P11的解离比TrpB2快，正如之前通过荧光法对分离出的P11所观察到的那样。因此，将肽与MalE的C末端融合可构成一种替代化学合成的方法，用于研究其在体内或体外被受体识别的情况。
