Results The small 13-amino-acid 11th strand of GFP (GFP11) was generated by PCR, annealing two phosphorylated oligonucleotides with appropriate restriction sites and cloning at the C-terminus of the D1.3 scFv gene in the pEP vector (Fig. 1). The His-tag at the C-terminus of GFP11 was used for purification by immobilized metal chromatography. The yield of scFv-D1.3-GFP11 was up to 5 mg per liter of bacterial culture. Binding activity comparison To determine whether fusion to the GFP11 peptide interferes with the expression and binding activity of the scFv, the scFv- D1.3-GFP11 molecule was compared to an essentially identical D1.3 scFv in fusion with the E-coil peptide [25], and lacking the GFP11 tag. Equal quantities of D1.3-E-coil and scFv-D1.3-GFP11 were used in an ELISA assay. The antibody antigen interaction was detected using SV5 antibodies and the scFv-D1.3-GFP11 was used with and without GFP1-10 incubation. The results were very similar for both constructs, at different concentrations, whether GFP1-10 was added or not, indicating that the GFP11 tag does not sterically hinder binding to the SV5 tag, even when the GFP molecule is fully complemented at the C-terminus of the scFv (Figure 2). Complementation assay In order to determine how effectively split GFP could be used to label a scFv molecule, we tested the complementation between serial dilutions of purified scFv-D1.3-GFP11 protein (75, 150, 300 pmol) and a molar excess of GFP1-10 (800 pmol). We measured the progression of fluorescence, indicating complementation, in a microtiter plate (Figure 3a). The fluorescent signal could be easily detected when illuminated at 488 nm within an hour after the addition of the complementary fragment GFP1-10 at room temperature with the fluorescence levels directly related to the different concentrations of scFv-D1.3-GFP11. In the absence of GFP1-10, there was no fluorescence. As protein purification is a time consuming process, we also carried out the assay using serial dilutions of a crude bacterial lysate. The curves obtained showed a significant increase in fluorescence after incubation with the GFP1-10 fragment, directly related to the scFv-D1.3-GFP11 dilution (Figure 3b). Quantification of scFv-D1.3-GFP11 using calibration curves In previous papers, GFP complementation using the GFP11 tag has been used to quantify proteins in E. coli [29] and eukaryotic cell extracts [32]. We assessed the ability to use the GFP11 tag to quantify scFvs in crude cell extracts using two standard curves prepared with complemented dilutions of sulfite-reductase GFP11 and purified scFv-D1.3-GFP11. Sulfite-reductase is the protein standard that we have previously used to calibrate GFP11 tagged proteins [30,31,33]. As shown in figure 4, there is a direct linear relationship between fluorescence and the concentration of SR- GFP11 and scFv-D1.3-GFP11, resulting in two almost superim- posable calibration curves. By measuring the fluorescence of a complemented sample of crude scFv-D1.3-GFP11 obtained from bacterial expression in deep well microtiter plates, and using the equation presented in Cabantous et al. [31], we were able to calculate the expression level of complemented scFv-D1.3-GFP11 under these conditions to be 5.6 mg/ml. This is approximately ten fold less than the amount of protein we routinely obtain from expression in large flasks, where aeration is signficantly better, and purification is probably more efficient. Split GFP Labeling of scFvs Figure 4. GFP1-10 standard curves to evaluate protein concentration. A GFP11 standard plot was constructed using increasing amounts of sulfite reductase GFP11 (SR-GFP11) and scFv- D1.3-GFP11 (ranging from 1.5 to 160 pmol) in a molar excess of GFP1- 10. The quantity of GFP11 tagged scFv in the cell extract was calculated according to the equations presented in Canbantou et al. [31]. In all cases fluorescence (a.u. arbitrary units) was measured after overnight incubation at 4uC. doi:10.1371/journal.pone.0025727.g004 Fluorescence linked immunosorbant assay (FLISA) Having shown that GFP11 was functional when fused to the C terminus of an scFv, we tested the antigen binding properties of the fluorescent scFv using a fluorescent linked immunosorbant assay [23], which exploited the fluorescence of the complemented GFP11. We have previously shown that FLISA is an effective and more rapid alternative to the use of ELISA to screen scFvs, when those scFvs are made fluorescent [23]. In particular, it is a single step assay that does not require additional incubations, beyond the addition of the fluorescently labeled antibody. Figure 5 shows that the assay has a very high signal to noise ratio when scFvs are fluorescently labeled using the split GFP system. An approxima- Figure 5. FLISA results at different complemented scFv-GFP11 concentrations. scFv-D1.3-GFP11 was used in a FLISA assay with binding between the scFv and its specific antigen assessed by complemented fluorescence. No signal was detected for the negative control myoglobin. doi:10.1371/journal.pone.0025727.g005 PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e25727