## 3 Results  
### The Dual Display of VNAR and GFP on the Surface of Yeast  
We utilized an altered yeast \(\alpha\)-agglutin display expression system to express the semi-synthetic yeast VNAR library on the surface of yeast. The display of fusion proteins mainly includes a VNAR followed by Aga2p, GFP, and Myc tag. To validate this expression system, we cloned a well-characterized lysozyme-binding VNAR (HEL-5A7) into a modified pNACP cloning vector (pNACP-GFP). When constructing pHEL5A7-GFP plasmids, the secretion signal peptide and HEL-5A7 VNAR gene were connected to the N-terminus of Aga2p via a GS (Glycine-Serine) linker, while the GFP and Myc tags were connected to the C-terminus of Aga2p to form the fusion protein gene (Figure 1A). A time-course experiment was performed to investigate the appropriate induction period after the pHEL5A7-GFP plasmid was transformed into EBY100 yeast cells. Figure 1B shows that after 24, 48, or 72 h of induction, the yeast can emit green fluorescence under a fluorescent microscope, indicating that the GFP protein at the C-terminus of the Aga2p fusion protein is expressed after being induced for at least 24 h. The GFP fluorescence intensity after induction was quantified by a fluorescent ELISA reader. Figure 1C shows that the GFP fluorescence from the unit of yeast cells increased with the prolonged induction time. The examination of GFP-positive cells by flow cytometry showed that after 72 h of induction, 54.2% of the yeast was GFP-positive cells (Figure 1D). These results indicated that the fusion protein is expressed in EBY100 yeast, but not certainly on the yeast surface. To verify whether the fusion protein is expressed on the surface of yeast, the anti-Myc antibody was used to detect the Myc tag at the C-terminus of the fusion protein in a cell-based ELISA. Since the conventional antibody is too large to enter the cells, if the anti-Myc antibody can detect the Myc tag at the C-terminus of the fusion protein, the target protein must be located on the surface of the yeast. The results of the cell-based ELISA, presented in Figure 1E, show that the induced yeast harboring pHEL5A7-GFP plasmid yielded a significant OD450 signal after being treated with different levels of the anti-Myc antibody. In contrast, the yeast-harboring pNACP-GFP plasmid had no obvious signal and it was revealed that after the plasmid pHEL5A7-GFP was transformed and induced for 72 h, the GFP of the fusion protein was correctly folded and transferred to the surface of the yeast. Next, we thought to demonstrate that VNAR fused to Aga2p and GFP could bind its antigen. The specific binding of the yeast surface's displayed HEL-5A7 fusion protein to lysozyme was verified by conducting a cell-based ELISA, a cell-based fluorescent assay, and a flow cytometry binding assay. Lysozyme or biotinylated lysozyme was co-incubated with the induced yeast, and the anti-lysozyme antibody or PE-conjugated streptavidin was used to detect the lysozyme bound to HEL-5A7. Figure 2A shows the results of the cell-based ELISA where lysozyme was detected by the anti-lysozyme antibody. As the lysozyme concentration increased, the OD450 nm of the yeast-harboring pHEL5A7-GFP also increased significantly, while the signal in the negative control group had no increment. In the experiment of biotinylated lysozyme detected by PE-conjugated streptavidin, the GFP signal was normalized to reveal the actual binding ratio of the GFP-expressing yeasts. As shown in Figure 2B, the resulting PE/GFP ratio increased with the biotinylated lysozyme concentration. The signal yielded by the lysozyme-binding yeast can prove that VNAR at the N-terminus end of Aga2p is correctly expressed and functional. Another cell-based fluorescent assay was conducted and investigated by flow cytometry. It was found that after the yeast bound with the biotinylated lysozyme, the proportion of GFP\({}^{+}\)PE\({}^{-}\) cells decreased from 70.03% to 39.33%, and the proportion of GFP\({}^{+}\)PE\({}^{+}\) cells increased from 0.01% to 21.09% (Figure 2C).