Muzard Julien, Adi-Bessalem Sonia, Juste Matthieu, Laraba-Djebari Fatima, Aubrey Nicolas, Billiald Philippe
Muséum national d'Histoire naturelle, CNRS FRE 3206-CP39, Paris cedex 05, France.
Anal Biochem. 2009 May 15;388(2):331-8. doi: 10.1016/j.ab.2009.02.035. Epub 2009 Mar 4.
Recombinant antibody fragments consisting of variable domains can be easily produced in various host cells, but there is no universal system that can be used to purify and detect them in the free form or complexed with their antigen. Protein L (PpL) is a cell wall protein isolated from Peptostreptococcus magnus, which has been reported to interact with the V-KAPPA chain of some, but not all, antibodies. Here we grafted the V-KAPPA framework region 1 (FR1) sequence of a high-affinity PpL-binding antibody onto single-chain antibody fragments (scFvs), which have no reactivity with PpL. This substitution made it possible to purify and detect scFvs using PpL conjugates. It did not hinder scFv folding and expression in recombinant bacteria, and it did not interfere with their antigen-binding function. We also identified residue 12 as being potentially able to alter PpL binding. This study, therefore, suggests a way of engineering a PpL-binding site on any scFv without interfering with its function. This could provide a universally applicable method both for the rapid purification of functional recombinant antibody fragments and for their detection even when complexed with their antigen without requiring fusion to an epitope Flag.
由可变结构域组成的重组抗体片段可以在各种宿主细胞中轻松产生,但目前还没有一种通用系统可用于以游离形式或与抗原复合的形式对其进行纯化和检测。蛋白L(PpL)是从大消化链球菌中分离出的一种细胞壁蛋白,据报道它能与部分(而非全部)抗体的Vκ链相互作用。在此,我们将一种高亲和力的PpL结合抗体的Vκ构架区1(FR1)序列嫁接到对PpL无反应性的单链抗体片段(scFv)上。这种替换使得利用PpL偶联物纯化和检测scFv成为可能。它不会阻碍scFv在重组细菌中的折叠和表达,也不会干扰其抗原结合功能。我们还确定第12位残基可能能够改变PpL结合。因此,本研究提出了一种在不干扰任何scFv功能的情况下构建PpL结合位点的方法。这可为功能性重组抗体片段的快速纯化以及即使在与抗原复合时(无需融合表位Flag)的检测提供一种普遍适用的方法。