Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
PLoS One. 2011;6(10):e25727. doi: 10.1371/journal.pone.0025727. Epub 2011 Oct 5.
Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we present an efficient method to fluorescently label single chain Fvs (scFvs) using the split green fluorescent protein (GFP) system. A 13 amino acid tag, derived from the last beta strand of GFP (termed GFP11), is fused to the C terminus of the scFv. This tag has been engineered to be non-perturbing, and we were able to show that it exerted no effect on scFv expression or functionality when compared to a scFv without the GFP11 tag. Effective functional fluorescent labeling is demonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore, we were able to show that this split GFP system can be used to determine the concentration of scFv in crude samples, as well an estimate of antibody affinity, without the need for antibody purification. We anticipate this system will be of widespread interest in antibody engineering and in vitro display systems.
抗体片段可从体外选择系统（如噬菌体和酵母展示）中轻易分离。由于缺乏抗体的 Fc 部分，通常使用抗体识别的小肽标签进行标记。在本文中，我们提出了一种使用绿色荧光蛋白（GFP）系统的有效方法来荧光标记单链 Fv（scFv）。将源自 GFP 最后一个β链的 13 个氨基酸标签（称为 GFP11）融合到 scFv 的 C 末端。该标签经过工程设计，不会干扰 scFv 的表达或功能，与没有 GFP11 标签的 scFv 相比没有任何影响。在许多不同的检测中，包括荧光联免疫吸附测定、流式细胞术和酵母展示，均证实了有效的功能性荧光标记。此外，我们还能够证明该 GFP 系统可用于确定粗样品中 scFv 的浓度以及抗体亲和力的估计值，而无需进行抗体纯化。我们预计该系统将在抗体工程和体外展示系统中得到广泛应用。
