AviTag fusion proteins used in the study.

Site specific biotinylation of AviTagged recombinant proteins using BirA enzyme is a widely used protein labeling technology. However, due to the incomplete biotinylation reactions and the lack of a purification method specific for the biotinylated proteins, it is challenging to purify the biotinylated sample when mixed with the non-biotinylated byproduct. Here, we have developed a monoclonal antibody that specifically recognizes the non-biotinylated AviTag but not the biotinylated sequence. After a ten-minute incubation with the resin that is conjugated with the antibody, the non-biotinylated AviTagged protein is trapped on the resin while the fully biotinylated material freely passes through. Therefore, our AviTrap (anti-AviTag antibody conjugated resin) provides an efficient solution for enriching biotinylated AviTagged proteins via a simple one-step purification.利用BirA酶对携带Avi标签（AviTag）的重组蛋白进行位点特异性生物素化，是目前应用广泛的蛋白质标记技术。然而，由于生物素化反应往往无法完全进行，且缺乏针对生物素化蛋白的特异性纯化手段，当样品中混杂未生物素化的副产物时，分离得到纯的生物素化样品极具挑战。本研究开发了一种单克隆抗体，可特异性识别未发生生物素化的AviTag序列，而不与已生物素化的该序列结合。将样品与偶联该抗体的树脂孵育10分钟后，未生物素化的带AviTag重组蛋白会被树脂捕获，而完全生物素化的蛋白则可自由流过该树脂。因此，本研究开发的AviTrap（抗AviTag抗体偶联树脂）可通过简便的一步纯化流程，高效富集生物素化的AviTag标记重组蛋白。