## 2 Materials and Methods
### Binding Assays for Yeast Clones against Lysozyme  
In the cell-based ELISA assay, 2 \(\times\) 10\({}^{7}\) induced yeast cells were washed with PBS once and incubated at room temperature for 1 h with 100 \(\upmu\)L of lysozyme (1% BSA in PBS). After washing three times using PBS, 100 \(\upmu\)L of a 1:5000 dilution of rabbit polyclonal anti-lysozyme antibody in PBS with 1% BSA was applied and incubated at room temperature for 1 h. Following three PBS washing steps, 100 \(\upmu\)L of a 1:20,000 dilution of HRP-conjugated \begin{table}
\begin{tabular}{l l l} \hline
**Primer Name** & **Sequence (5\({}^{\prime}\) to 3\({}^{\prime}\))** & **Comments** \\ \hline For-1 st & GCTCGAGTGGCACCAAACCGC & Designed in this study \\ \hline \multirow{2}{*}{Rev12-X3 CX2 CX5} & CGCGACGTCCATCTCCGCAAMNNMN- \\  & NMNNMCAMNNMNACAMNNMN- \\  & MNNMNNMNGAGACCGCAACGTGTCCAC \\ \hline \multirow{2}{*}{Rev13-X6 CX1 CX4} & CGCGACTGTCCGCAMNNMN- \\  & NMNNNNMNMNNNGCAMNNNACA- \\  & MNNMNNMNMNNNGAGACCGCAAACGTGTCCAC \\ \hline \multirow{2}{*}{Rev16-X6 CX3 CX5} & CGCGACGTCCATCTCTCCGAAMNNMN- \\  & NMNMNNNNMNNNGCAMNNMN- \\  & NMNNNNNACAMNNNNMNMN- \\  & NMNNNNNGAACCGCAAACGTACCACC \\ \hline \multirow{2}{*}{Rev20-X5 CX5 CX8} & CGCGACGTCCATCTCCCCAMNNMN- \\  & NMNNNNMNNGCAMNNNNMN- \\  & NMNNNNNGAACCGCCAACGTACCACC \\ \hline \multirow{2}{*}{Rev20-X5 CX5 CX8} & CGCGACGTCCATCTCCCCAMNNMN- \\  & NMNNNNMNNGCAMNNNNMNMN- \\  & NMNNNNNGAACCGCCA- \\  & ACGATACGTCCCAC \\ \hline \multirow{2}{*}{Rev23-X6 CX5 CX10} & CGCGACTGTCCGCAMNNMN- \\  & NMNNNNNNMNACAMNNNNMN- \\  & NMNNNNNNNGACCGAACGTCCAC \\ \hline \multirow{2}{*}{For-TU1 mod-40 fl} & GCTAAAAAGAAGGGGTACAAATTAA- \\  & GATAAAAAAGAGGGCCAAGCTGT- \\  & CGAGTGGCACAAACAC \\ \hline \multirow{2}{*}{Rev-TU58 mod-40 fl} & AGCACCACCACCCTCTGATCC- \\  & ACCAGACCACCACCCTCCGATCCATTCC- \\  & ACAGTCACGGCCATCTCCCCATCTCC \\ \hline \end{tabular}
\end{table} 
Table 1: Primers used in this study. goat anti-rabbit IgG (H + L) in PBS with 1% BSA was applied and incubated for 1 h at room temperature. Subsequently, the cells were washed three times and incubated with 100 \(\upmu\)L stabilized chromogen (Thermogisher, catalog: SB01). The reaction was stopped by adding 100 \(\upmu\)L of stop solution (Thermogisher, catalog: SS03100). The absorbance of cell-free supernatant was measured at 450 nm by BioTek Epoch ELISA reader (Bio-Tek Instruments, Inc., USA). In the cell-based fluorescent assay, 2 \(\times\) 10\({}^{7}\) induced yeast cells were washed with PBS once and incubated at room temperature for 1 h with 100 \(\upmu\)L of biotinylated lysozyme (1% BSA in PBS). After washing three times using PBS, 100 \(\upmu\)L of a 1:100 dilution of PE-conjugated streptavidin in PBS with 1% BSA was applied and incubated at room temperature for 1 h. Subsequently, the cells were washed three times and resuspended with PBS. These cells were added into a 96-well microwells plate (Corning, catalog: 3916), then the GFP and PE fluorescence intensity were measured by a fluorescent ELISA reader (SpectraMax Gemini XPS/EM, Molecular Devices, San Jose, CA, USA). The PE to GFP ratio is calculated by dividing PE readings by GFP readings. In the flow cytometry binding assay, 5 \(\times\) 10\({}^{7}\) induced yeast cells were washed with PBS once and incubated with biotinylated lysozyme (1% BSA in PBS) at room temperature for 1 h. After washing three times using PBS, 100 \(\upmu\)L of a 1:100 dilution of PE-conjugated streptavidin in PBS with 1% BSA was applied and incubated at room temperature for 1 h. The cells were washed three times and resuspended with PBS for subsequent flow cytometry analysis by Moflot(tm) XDP high-performance cell sorter (Beckman Coulter, Brea, CA, USA). The data were analyzed using Summit software ver. 5.3 (Summit Software, Little Rock, AR, USA). At least 3.5 \(\times\) 10\({}^{5}\) yeast cells per sample were analyzed and grouped based on discrimination of GFP and PE fluorescence intensity, representing those yeast cells displaying VNAR on the surface and binding biotinylated lysozyme.