Wittrup: Yeast surface display
1. Induce at least a 10-fold excess of library size of freshly grown cells in SGCAA at a starting OD600 of ~0.5-1. Induce at 20 degC for 20-24 h with shaking at 250 rpm. Induction of cells during exponential growth (OD600 2-5) has been shown to increase scFv expression. The addition of 2 g/L dextrose to SGCAA induction media can also improve surface display levels. Induction temperature can be raised to 30 degC or 37 degC to select for more stable clones.
2. Pellet an appropriate number of induced cells at 12,000 rpm for 30 s in a 1.5-mL microfuge tube, aspirate the supernatant, and wash with 1 mL PBS/BSA buffer (rinse, re-pellet, and aspirate supernatant). If labeling a library population for FACS selection, label a 10-fold excess of population diversity. For characterization of a population or clone, it is convenient to work with a quantity of cells that forms a visible pellet upon centrifugation, usually 10^6 cells.
3. Label yeast with chicken anti-c-myc IgY (1:250) and an appropriate concentration of biotinylated antigen in an appropriate final volume of PBS/BSA. Vortex to resuspend. Typical labeling volumes are 50 uL for 10^6 cells and 0.5-1 mL for 10^8 cells. The labeling volume must be large enough to allow yeast to stay in suspension and should be chosen such that antigen binding to yeast-displayed scFv is not under depleting conditions. This can be accomplished by maintaining at least a 10-fold molar excess of antigen over scFv. Maintaining a 10-fold antigen excess may not be important in early rounds of sorting, where many scFv clones may not bind antigen to a large degree. However, as the scFv affinity increases, the 10-fold molar excess becomes essential. The sample can also be incubated with rotation to keep cells in suspension.
4. Incubate cells at room temperature for an appropriate amount of time (usually 30 min, but can be up to 3 h). Resuspend cells as necessary during incubation.
5. Pellet cells at 12,000 rpm for 30 s at 4 degC and wash with 1 mL ice-cold PBS/BSA. This and all subsequent steps for yeast labeling should use ice-cold PBSF buffer and be performed on ice or at 4 degC.
6. Label yeast with appropriate secondary reagents, e.g. goat anti-chicken IgG-Alexa488 (1:100) and streptavidin-PE (1:100), in an appropriate final volume (usually 50 uL for 10^6 cells). Vortex cells to resuspend. Typical dilutions for other secondary reagents are neutravidin-PE (1:50) and streptavidin-APC (1:100). A working dilution for other secondary reagents should be determined by titration.
7. Incubate cells on ice and shielded from light for 10-20 min. For the above reagents, a significant fluorescence signal is seen after 5 min of incubation. Longer incubation may increase the signal, but is balanced by the rate at which antigen dissociates from the scFv.
8. Pellet cells for 12,000 rpm for 30 s at 4 degC and wash with 1 mL PBS/BSA. Keep cell pellet on ice.
9. Prior to analysis, set up a FACS protocol using control yeast samples. One should prepare secondary-only, Alexa488-only, and PE-only control samples. The yeast population is normally gated on forward and side scatter channels to remove debris and aggregated cells (usually <5% of the sample). In addition, the FACS machine should be properly compensated to reject crosstalk between the Alexa488 and PE channels of the fluorescence detector, as compensation is necessary when there is a significant overlap between the emission spectra of the two fluorphores.
10. Resuspend cell sample in PBS/BSA directly prior to FACS analysis. The volume for the XL should be at least 500 uL, and the cell concentration should not exceed 10^8/mL for sorting. Load the cell sample into the FACS machine.
