In my lab, we are engineering next generation cellular therapies by combining protein engineering with cellular engineering. So we engineer protein components to obtain new functions or to improve existing ones, and then incorporate these null proteins into cellular therapeutics. We have further developed use surface display for specialized applications including protein stability engineering, engineering of small-molecule regulated protein switches, and specific targeting of cancer associated activated receptor confirmations.
The advantage of yeast surface display compared to other display technologies such as phage or ribosome display, is the flow cytometric visualization of the library during the sorting process and the precise control of the selection conditions, such as antigen concentration and stringency of the sorting gate. To begin, prepare the beads for the first bead selection by resuspending 10 microliters of biotin binder magnetic beads in 990 microliters of PBSA. For washing place the tube on a magnetic rack for two minutes with the lid open.
Then carefully remove the supernatant. Resuspend the washed beads in a 1.5 milliliter micro centrifuge tube in one milliliter of PBSA with 6.7 to 33 picomoles of biotinylated antigen. After incubation, place the tube on a magnetic rack for two minutes with the lid open, remove the supernatant and wash the antigen loaded beads with one milliliter of PBSA.
Once the beads settle, remove the PBSA. Then re suspend the antigen loaded beads in 50 microliters of PBSA. Prepare the cells, antigen beads, and a solution of bear beads for negative selections.
After washing resuspend the antigen beads in 50 microliters of PBSA and resuspend the bear beads in 150 microliters of PBSA. For the first negative selection, add 50 microliters of washed bare beads to 950 microliters of washed cells in PBSA and incubate the mixture for 1.5 hours at four degrees celsius. After the incubation, place the tubes containing the bear bead cell suspensions on a magnetic rack with the lid open.
Pipette any liquid in the lid into the tube, and wait for two minutes. Then transfer the unbound cells to a fresh micro centrifuge tube and add 50 microliters of washed bear beads. After three rounds of negative selection, add 50 microliters of antigen-loaded bead solution to the cells.
Incubate for two hours at four degrees Celsius. Now place the cells containing the antigen-loaded beads on a magnetic rack with the lid open. Pipette any liquid that is in the lid into the tube.
Wait for two minutes Before discarding unbound cells. Perform all remaining steps as described for the first antigen bead selection. After three negative and one positive selection, resuspend cells rapidly in one milliliter of SDCAA and transfer to a shaker flask containing a larger volume of SDCAA.
After overnight induction of surface expression of proteins in SGCAA in yeast libraries, pellet enough cells to cover 10 times the diversity and discard the supernatant. Resuspend the pellet in PBSA and transfer it to micro centrifuge tubes. Use 30 million cells for staining in each tube.
Prepare as many tubes as required based on the diversity, including one control tube without the antigen. Then centrifuge the tubes at 2000 x g for five minutes at room temperature. Resuspend the pellet in 200 microliters of PBSA containing the antigen, and incubate for one hour at four degrees Celsius.
After performing the centrifugation once again, remove PBSA from the pellet. Next resuspend the cells in 100 microliters of cold PBSA containing antibodies for display staining and antigen detection. Incubate for 30 minutes at four degrees Celsius.
After incubation, centrifuge the cells at 2000 x g for five minutes at four degrees celsius. Add one milliliter of PBSA to the pellet and repeat the centrifugation. Then remove most of the supernatant, leaving only 20 to 30 microliters to prevent the pellet from drying out.
Resuspend the pellet in cold PBSA right before sorting. Now sort the cells directly into SDCAA a medium. After the sort, transfer cells to a shaker flask containing a larger volume of SDCAA medium and incubate at 30 degrees celsius with shaking at 180 RPM.
