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.
Highlights
Monoclonal antibodies have long been used for biological and medical research, as well as in vitro diagnostics
A number of publications [15,16,17] deal with the creation of such fluorescent antibody fragments, the yields have been very disappointing, mainly due to the fact that single chain Fvs (scFvs) contain disulfide bonds that require oxidizing environments for correct folding, while green fluorescent protein (GFP) folds in the reducing cytoplasm and not in the periplasm [18]
The His-tag at the C-terminus of GFP11 was used for purification by immobilized metal chromatography
Summary
Monoclonal antibodies (mAbs) have long been used for biological and medical research, as well as in vitro diagnostics. A number of publications [15,16,17] 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
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