Yeast surface display (YSD) is a powerful technique for expressing proteins, peptides, or antibody fragments on the surface of yeast cells, allowing researchers to screen and study protein-protein, protein-peptide, or protein-small molecule interactions. The following protocol outlines the steps for constructing and using a yeast surface display system.

 Yeast Surface Display Protocol

 Materials Needed

1. Yeast strain: _Saccharomyces cerevisiae_ strain (commonly EBY100, which has been optimized for surface display).
2. Yeast expression vector: Plasmid vector (e.g., pCTCON2 or pYD1) with the gene of interest cloned in frame with the surface display protein (e.g., Aga2p).
3. Growth media:

SD-CAA (minimal selection media): For plasmid selection.

SG-CAA (induction media): For induction of protein expression (galactose-containing media).

4. Enzymes and reagents:

DNA extraction kits or methods.

Restriction enzymes for cloning.

5. Antibiotics: For selection, if applicable.
6. Fluorescently labeled antibodies/target molecules: Used for detection of displayed proteins.
7. PBS: Phosphate-buffered saline for washing steps.
8. Induction buffer: To induce protein expression in yeast.
9. Plasmid DNA preparation kits.

 Equipment Needed

1. Shaking incubator: For growing yeast cultures at optimal temperatures.
2. Centrifuge: For pelleting cells.
3. Flow cytometer (FACS): For sorting yeast cells based on surface display and target binding.
4. Microcentrifuge tubes: For handling small volumes of yeast cultures and reagents.
5. Electroporator or chemical transformation materials: For yeast transformation.

Protocol

Cloning the Gene of Interest into a Yeast Surface Display Vector

Design the gene of interest: The gene encoding the protein or peptide of interest should be cloned into the yeast display vector (e.g., pCTCON2) in frame with the surface anchor protein gene, such as Aga2p.

The vector typically contains a galactose-inducible promoter (GAL1) for controlled expression.

Restriction digestion and ligation: Use appropriate restriction enzymes to cut the vector and the insert (gene of interest), followed by ligation to create the expression construct.

Transformation of E. coli: Transform the ligated plasmid into E. coli for amplification and plasmid preparation. Isolate the plasmid DNA using a DNA preparation kit.

Verification: Sequence the plasmid to verify correct insertion and reading frame with the Aga2p gene.

Transformation of Yeast Cells

Prepare yeast cells: Use _Saccharomyces cerevisiae_ (such as EBY100) as the host strain.

Transform the plasmid into yeast:

Chemical transformation: Use the lithium acetate method or electroporation to transform the plasmid into yeast cells.

Incubate on SD-CAA plates (synthetic defined medium with casein amino acids) that lack uracil or another selectable marker, to select for yeast cells that have taken up the plasmid.

Colony isolation: Grow transformed yeast cells on selective plates at 30°C for 2-3 days until colonies form.

Culture and Induction of Protein Expression

Inoculation and culture:

Inoculate a single colony from the transformation plate into SD-CAA medium and grow overnight at 30°C with shaking (250–300 rpm).

Induction of protein expression:

The next day, pellet the yeast cells by centrifuging at 1,000–3,000 x g for 5 minutes.

Wash cells with sterile water or PBS and resuspend them in SG-CAA medium (containing galactose) to induce the expression of the protein on the yeast surface.

Incubate at 20°C or 30°C for 24–48 hours with shaking to allow for protein display.

Confirmation of Protein Display

Sample preparation: After induction, collect 1 mL of the culture and pellet the yeast cells by centrifugation (1,000–3,000 x g for 5 minutes).

Washing: Wash the cells twice with cold PBS to remove residual media and prevent non-specific binding in downstream assays.

Fluorescent labeling:

Incubate the yeast cells with a primary antibody or fluorescently labeled target molecule that binds to the protein of interest. If using a primary antibody, follow with a secondary fluorescently labeled antibody.

Typically, 30 minutes to 1 hour at 4°C is sufficient for incubation, with gentle mixing to avoid settling of yeast cells.

Washing: After labeling, wash the cells 2-3 times with PBS to remove unbound antibodies or fluorescent probes.

Flow cytometry: Analyze the labeled yeast cells using a flow cytometer (FACS). The presence of fluorescence indicates successful display of the protein on the yeast surface.

Screening for High-Affinity Binders (Optional)

If you are screening for high-affinity binders, such as in antibody discovery or protein engineering:

Incubate yeast cells with target: Incubate the yeast cells with the fluorescently labeled target molecule (e.g., an antigen, protein, or small molecule).

This step helps identify yeast cells displaying proteins or peptides that specifically bind to the target molecule.

FACS sorting: After incubation, use FACS to sort yeast cells that show strong binding to the target (high fluorescence). Collect the sorted cells for further rounds of selection and enrichment.

Iterative rounds: Perform additional rounds of growth, induction, and FACS sorting to progressively enrich for yeast cells displaying proteins or peptides with higher affinity for the target molecule.

Recovery and Sequence Analysis

DNA extraction: After sorting, extract plasmid DNA from the enriched yeast cells to identify the sequences of the selected binders.

Yeast plasmid DNA can be extracted using yeast-specific plasmid prep kits or protocols.

Transformation into E. coli: Transform the extracted plasmid DNA into E. coli for amplification and sequencing.

Sequence analysis: Sequence the plasmids to identify the mutations or variants that led to enhanced binding or functional properties.

Characterization of Selected Variants

Once specific yeast clones have been isolated, express the proteins or peptides in larger quantities for further biochemical and functional characterization:

Binding affinity measurement: Use surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to measure the binding affinity of the selected proteins.

Functional assays: Perform any necessary functional assays to validate the biological activity of the selected proteins or peptides.

 Important Considerations

Glycosylation: Yeast cells are eukaryotic, meaning they glycosylate proteins. This is advantageous for many applications, but yeast glycosylation patterns differ from mammalian systems, which can sometimes affect the biological activity of the displayed proteins.

Temperature for induction: Lower temperatures (20°C) may help improve proper protein folding and display efficiency, especially for complex or large proteins.

Antigen/target concentration: Use an optimized concentration of the target molecule to avoid non-specific binding or saturation during sorting or affinity screening.

 Conclusion

Yeast surface display is a powerful tool for protein engineering, antibody discovery, and studying molecular interactions. This protocol provides the steps to construct, express, and screen proteins or peptides on the surface of yeast cells, making it useful for applications ranging from high-affinity binder discovery to functional protein analysis.