Chatellier J, Rauffer-Bruyère N, Van Regenmortel M H, Altschuh D, Weiss E
Institut de Biologie Moléculaire et Cellulaire du CNRS, Laboratoire d'Immunochimie, Strasbourg, France.
J Mol Recognit. 1996 Jan-Feb;9(1):39-51. doi: 10.1002/(sici)1099-1352(199601)9:1<39::aid-jmr239>3.0.co;2-v.
Two recombinant Fab fragments, 57P and 174P, recognizing peptide 134-146 of the coat protein of tobacco mosaic virus have been cloned, sequenced and expressed in Escherichia coli. They differ by 15 amino acid changes in the sequence of their variable region. The interaction kinetics of the Fabs with the wild-type and four mutant peptides have been compared using a BIAcoreTM biosensor instrument. The recombinant Fab 174P had the same reactivity as the Fab fragment obtained by enzymatic cleavage of monoclonal antibody 174P. The two recombinant Fabs recognized the various peptides in the same ranking order but Fab 174P consistently dissociated somewhat faster from the peptides compared to Fab 57P. The two whole antibodies showed the same relative differences in reactivity as the two recombinant Fabs. The location of amino acid changes was visualized on a model structure of the Fab. Differences in dissociation rates of the two antibodies are most likely due to changes located at the periphery of the antigen-combining site and/or at the interface between the light and heavy chain domains. Our results demonstrate the feasibility of detecting very small differences in binding affinity by the biosensor technology, which is a prerequisite for assessing the functional effect of limited structural changes.
已克隆、测序并在大肠杆菌中表达了两种识别烟草花叶病毒外壳蛋白第134 - 146位肽段的重组Fab片段57P和174P。它们可变区序列有15个氨基酸的差异。使用BIAcoreTM生物传感器仪器比较了这些Fab片段与野生型及四种突变肽段的相互作用动力学。重组Fab 174P与通过单克隆抗体174P酶切获得的Fab片段具有相同的反应性。两种重组Fab以相同的排序识别各种肽段，但与Fab 57P相比，Fab 174P从肽段上的解离始终稍快一些。两种完整抗体在反应性上表现出与两种重组Fab相同的相对差异。在Fab的模型结构上直观显示了氨基酸变化的位置。两种抗体解离速率的差异很可能是由于位于抗原结合位点周边和/或轻链与重链结构域之间界面处的变化所致。我们的结果证明了利用生物传感器技术检测结合亲和力非常小的差异的可行性，这是评估有限结构变化的功能效应的一个先决条件。
