Enzymatic Modification and Flow Cytometry Assessment of Yeast Surface Displayed Proteins

This research focuses on studying the chemical modifications to proteins that dictate how cells behave. In this case, the goal is to understand the preferences and kinetics of enzymes modifying a variety of substrates.

The ability to dephosphorylate a substrate molecule displayed on the surface of yeast has been demonstrated for the first time. This finding opens possibilities for generalizable kinetic mapping of enzymatic modifications that were previously not accessible. This research addresses the challenges associated with understanding enzyme substrate interactions. Typically, cell lysates or customized peptides are required for this type of assessment. Here, a FasL alternative is provided through the use of yeast-displayed substrates.

This protocol advances traditional yeast surface display towards analysis of what would ordinarily be intracellular enzymatic reactions in the extracellular space, enabling FasL analysis of these normally difficult-to-assess interactions.

This protocol enables the ability to interrogate substrate preferences of a variety of enzymes through high-throughput methods and to perform kinetic studies with a simple readout.

[Instructor] To begin, thaw an aliquot of the prepared yeast cells on ice. Add 0.5 to 1.5 micrograms of plasmid DNA containing the yeast display construct directly to the cells. Then add 0.5 milliliters of either the transformation solution or a sterile 50% polyethylene glycol and 0.1 molar lithium acetate solution. Thoroughly combine the mixture of cells, plasmid DNA and transformation solution by pipetting. Incubate the transformation mixture statically for 30 to 60 minutes at 30 degrees Celsius, and vortex the mixture at 15-minute intervals. Harvest the cells by centrifuging at 1000G. Prepare a 14-milliliter culture tube with 4.5 milliliters of plasmid-containing yeast cell growth, or SDCAA media. Re-suspend the cells containing the desired plasmid in 500 microliters of SDCAA and inoculate the prepared 4.5 milliliters of media. Distribute 50 microliters of the five milliliters of inoculated culture onto an SDCAA plate carefully without piercing the auger. Incubate statically at 30 degrees Celsius for 48 hours to determine transformation efficiency. Now, incubate the five milliliters of SDCAA cell culture in a shaking incubator at 30 degrees Celsius and 300 RPM for at least 18 hours. Monitor optical density at 600 nanometers after 16 hours, and repeat again after 20 hours. Once the sample reaches an optical density not exceeding six, centrifuge the culture for three minutes at 2500G, and discard the supernatant without disturbing the yeast pellet. Re-suspend the yeast pellet in the induction of protein expression, or SRGCAA media, to a final optical density at 600 nanometers less than one. Incubate the yeast culture in a shaking incubator at 30 degrees Celsius and 300 RPM for at least eight hours, but no longer than 24 hours. Then measure the optical density at 600 nanometers to determine the cell density. Prepare the working buffer for the samples by diluting the 2X working buffer solution in a one-to-two ratio with deionized water in a 1.7-milliliter vial. Add the appropriate volume of yeast culture required to recover two million yeast cells into the 1.7-milliliter vial for each sample. Centrifuge the vial for one minute at 4500G, and using a micro pipette, carefully remove and discard the supernatant as biohazardous waste. Re-suspend the pelleted cells in one milliliter of PBSA before repeating the centrifugation and supernatant removal. Keep it aside. Prepare the recombinant human SHP-2 solution at a final concentration of 1,000 nanomoles in a 20-microliter total reaction volume. Next, add 7.7 milligrams of DTT into 10 milliliters of deionized water in a 15-milliliter conical tube to create a five-millimolar DTT solution. Now, take the pelleted cells and re-suspend them in the working buffer so that the final reaction volume is 20 microliters per sample. Add two microliters of the five-millimolar DTT solution to each sample for a final DTT concentration of 0.5 millimolar. Add the prepared volume of SHP-2 to each sample to achieve a final volume of 20 microliters, and gently mix using a micro pipette. Wrap the sample vial lids with Parafilm to prevent leakage or cross-contamination. Incubate the samples at 37 degrees Celsius for two hours on a rotor at a constant speed. After removing the samples from the rotor, stop the reaction by adding one milliliter of PBSA to each sample before centrifuging them again for one minute. Then discard the supernatant. Re-suspend the pelleted samples in a 20-microliter mix of their corresponding primary reagents, and incubate for 20 minutes at room temperature. After 20 minutes, centrifuge samples at 4500G for one minute and discard the supernatant as biohazardous waste. Wash the cells once by re-suspending them in one milliliter of PBSA before centrifuging the cells again. Now, re-suspend the samples in a 20-microliter mix of their corresponding secondary reagents, and incubate for 15 minutes in the dark. After centrifuging the samples and discarding the supernatant, wash the cells once more with PBSA before centrifuging them again. Next, re-suspend the washed samples in 300 to 500 microliters of PBSA, and transfer them to five-milliliter polystyrene tubes for flow cytometry analysis. After preparing the cytometer, click on File, followed by the New Experiment button, name the experiment and click Save to ensure the data acquired is saved in the desired file path. Select the dot plot icon in the upper toolbar to create two or more dot plots for each sample. For one of the dot plots, ensure the x-axis name displays the FSCA channel, and the y-axis name displays the SSCA channel. For another plot, select the x-axis name to display the channel in which the secondary reagent targeting the primary anti-epitope tag antibody fluoresces, and the y-axis name to display the channel in which the streptavidin secondary reagent fluoresces. Provide a descriptive name for the sample by right-clicking on the tube, selecting Edit Name and entering the sample name. Place each sample tube in the holder of the cytometer, and click Run to begin loading the sample and acquiring data. Adjust events, time and flow rate as necessary. Define a gate surrounding the healthy yeast cells in the SSCA versus FSCA plot. Then record the fluorescence of all control samples. Define a gating strategy for the created plot before analyzing the treated samples. Record the fluorescence of dephosphorylated samples using the cytometer and the gating strategy before analyzing the data using flow cytometry software. Evaluate phosphorylation by measuring and comparing the y-axis median of cells expressing protein on their surface, and the baseline phosphorylation provided by non-playing cells between samples and controls. Finally, calculate the percent median phosphorylation difference using the provided formula. The effect of varying SHP-2 phosphatase concentrations and incubation times on the median phosphorylation difference in yeast surface-displayed substrates is illustrated in this figure. Percent median phosphorylation difference decreased over time across all SHP-2 concentrations, with the highest dephosphorylation observed at 1,000 nanomolar SHP-2 concentration at four hours`. After two hours of incubation, 1,000 nanomolar SHP-2 resulted in approximately 48.8% median phosphorylation difference, significantly lower than the 750 nanomolar condition at the same time point,. No significant difference in median phosphorylation difference was observed when incubation was extended from two hours to three hours across 500-nanomolar, 750-nanomolar and 1,000-nanomolar SHP-2 concentrations.