Fluorescent Labeling of Antibody Fragments Using Split GFP Fortunato Ferrara, Pawel Listwan, Geoffrey S. Waldo, Andrew R. M. Bradbury* Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America Abstract 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. Citation: Ferrara F, Listwan P, Waldo GS, Bradbury ARM (2011) Fluorescent Labeling of Antibody Fragments Using Split GFP. PLoS ONE 6(10): e25727. doi:10.1371/ journal.pone.0025727 Editor: Dafydd Jones, Cardiff University, United Kingdom Received March 24, 2011; Accepted September 9, 2011; Published October 5, 2011 Copyright: 2011 Ferrara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funded by the NIH (P50GM085273 and R01-HG004852-01A1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: amb@lanl.gov Introduction Monoclonal antibodies (mAbs) have long been used for biological and medical research, as well as in vitro diagnostics. However, the derivation of mAbs is relatively expensive and time consuming. Over the past decade, an alternative and effective way to obtain monoclonal antibodies has been the use of recombinant antibody display libraries from which antibodies of interest can be selected and expressed in E. coli [1]. Filamentous phage display [2] is the most widely distributed display platform, with yeast [3] and ribosome display [4] also frequently used. The commonest antibody formats used are single chain Fvs (scFvs) [5] and Fabs. scFvs are recombinant constructs in which the variable domains of the heavy and light chains are covalently linked together by a flexible linker [5], whereas Fabs were originally described by the digestion of antibody with papain [6], and consist of two protein fragments, VH-CH1, and VL-CL that can be expressed separately in the E. coli periplasm where they assemble together [7]. Full- length antibodies include the Fc region, which, from a reagent point of view, can be considered to be a large tag recognized by secondary antibodies or bacterial superantigens, such as protein A or G. Antibody fragments, in contrast, are usually modified by the fusion of in-frame tags to the antibody fragment. The commonest format includes a short peptide tag (e.g. myc [8] or SV5 [9]) recognized by a monoclonal antibody, and a histidine tag that can be used for purification by immobilized metal affinity chromatog- raphy [10]. Detection of antibody fragments using peptide tags generally requires an extra step when compared to the detection of full-length antibodies, since anti-peptide and subsequent labeled secondary antibodies are usually required. Given the recombinant nature of antibody fragments selected from phage display libraries, it was initially thought it would be relatively trivial to fuse effector domains directly to antibody fragments. Direct fusion to alkaline phosphatase has been extremely successful [11–14], with scFv-AP fusions being relatively well expressed and stable, allowing direct enzyme linked immunosorbant assays (ELISAs) to be carried out without the need for additional reagents. In a similar manner, covalent fusion to green fluorescent protein (GFP) or similar proteins should allow fluorescent labeling of scFvs. This would also provide the significant advantage that each scFv would be labeled with only one fluorophore, potentially allowing scFv quantification by fluorescence. Although a number of publications [15–17] deal with the creation of such fluorescent antibody fragments, the yields have been very disappointing, mainly due to the fact that scFvs contain disulfide bonds that require oxidizing environments (such as eukaryotic secretory pathways, or the bacterial periplasm) for correct folding, while GFP folds in the reducing cytoplasm and not in the periplasm [18]. This is in contrast to alkaline phosphatase, which folds in the same oxidizing environments as antibody fragments, explaining the greater success attained with this fusion. Recently, a number of reports [19–21] have described split GFP assays, in which self-complementing fragments of GFP are non- fluorescent individually, but recreate functional GFP when present together. One of these systems, in particular [19], is based on a small 13 amino acid fragment corresponding to the 11th strand of GFP (GFP11) that was evolved to have minimal effect on the function or solubility of the protein to which it is fused. This peptide can restore fluorescence to an evolved version of the first 10 strands of GFP (GFP1-10) with which it interacts almost irreversibly. A potential advantage of tagging with the small split PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e25727