GFP is that the protein of interest can complete folding without the interference of a full-length GFP attached. Furthermore, the small split GFP tag is less likely to interfere with normal transport to the periplasm. In this study, we described the genetic fusion of the GFP11 peptide to the well-characterized anti-chicken-lysozyme scFv, D1.3, and its expression and functionality after expression in E. coli. The utility and detectability of the recombinant antibody was evaluated in a fluorescence linked immunosorbant assay (FLISA) [17,22,23], an immunosorbant assay that uses fluorescence rather than enzymatic activity for detection, in yeast display, and in a multiplex flow cytometry assay for high-throughput purposes. Materials and Methods DNA constructs The GFP11 Fragment was created by annealing the two oligonucleotides GFP11-rev (P – GAT CCG CCA CCT GTA ATC CCA GCA GCA TTT ACG TAC TCA TGA AGG ACC ATG TGG TCA CGA GT) and GFP11-for (P – ACT CGT GAC CAC ATG GTC CTT CAT GAG TAC GTA AAT GCT GCT GGG ATT ACA GGT GGC G). This creates the GFP11 fragment with restriction site ends (ScaI and BamHI) compatible Split GFP Labeling of scFvs with the pEP-D1.3 vector [24] (a pET based scFv expression vector, in which an E coil [25] is in fusion with the scFv) (Figure 1a). The final construct consists of the D1.3 scFv fused to GFP11and a His tag at the C terminus (Figure 1b–c), with the E- coil removed. In order to generate the yeast display vector, pDNL6-GFP11, the D1.3-GFP11 fragment was PCR amplified using a modified GFP11-forward primer that replaces the BamHI restriction site with NotI. Purified fragments were digested with BssHII and NotI and cloned into pDNL6, a yeast display vector based on pPNL6 [26] which contains BssHII and NheI as cloning sites in order to be compatible with our phage display vector, pDAN5 [27] (Figure 1a). Antigen preparation Neutravidin (Pierce Inc.), Lysozyme and Myoglobin, were purchased (SIGMA Aldrich), while IgE receptor was donated by Graham Mackay, University of Melbourne. All antigens were biotinylated using the sulfo NHS biotinylation system (Pierce Inc.) according to the manufacturer’s instructions. The purified anti- SV5 antibody was conjugated with alkaline phosphate enzyme (AP) using the Lightning-LinkTM AP Conjugation Kit (Innova Biosciences) according to the manufacturer’s instructions. Figure 1. Split-GFP labeling of scFvs. A. GFP complementation vectors. The scFv-D1.3-GFP11 construct was cloned into a pEP based vector (left) or a yeast display vector (right). The GFP11 peptide is cloned at the C-terminus of the scFv in order not to interfere with the scFv/antigen- binding activity. B. Linear representation of the scFv-GFP11 molecule. The sequence of the SV5 tag, followed by the GFP11 sequence and the six histidine tag (6xHis tag) found at the C terminus of the scFv is shown. C. Split GFP fragment complementation. The scFv is fused to the small GFP fragment (strand 11, residues 215-230). The complementary GFP fragment (1-10, residues 1-214) is expressed separately. Neither fragment alone is fluorescent. When mixed, the small and large GFP fragments spontaneously associate, resulting in reconstitution of the fluorophore and fluorescence. D. Split GFP fragment complementation in scFv yeast display. The scFv in fusion with the GFP11 strand is displayed on yeast cells. It is still able to bind the specific antigen and fluorescence is restored when yeast cells are incubated with the GFP1-10 fragment. doi:10.1371/journal.pone.0025727.g001 PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e25727