Enzymatic Modification and Flow Cytometry Assessment of Yeast Surface Displayed Proteins

Summary

Here, we introduce a protocol for using yeast surface-displayed substrates for enzymatic modification assays. The platform was demonstrated using the analysis of the dephosphorylation activity of tyrosine phosphatase SHP-2 against one of its substrates as a representative enzymatic modification assay.

Abstract

Yeast surface display is a genotype-phenotype linkage strategy that empowers high-throughput screening of protein function. Traditionally, yeast surface display has been applied to the evolution of new binding proteins, with flow cytometry used to assess and sort by levels of binding strength. Recently, there has been growing interest in applying yeast surface display for screening enzymatic modification of substrate variants, with additive (e.g., phosphorylation) or subtractive (e.g., proteolysis) modifications providing a phenotype readable by flow cytometry. Such modifications are regularly applied using intracellular co-localization, but the ability to achieve extracellular enzymatic modification of displayed substrates could open many more reactions to investigation. Here, we describe techniques for designing and applying screening assays for extracellular enzymatic modification to candidate substrates displayed on the yeast surface and subsequent evaluation using flow cytometry analysis. We provide these protocols in the context of phosphatases dephosphorylating yeast displayed substrates containing phosphorylated tyrosine residues and comment on how this applied framework can be adapted to developing screening assays for other enzyme-substrate pairs.

Introduction

The understanding of the interactions between enzymes and their targets has become an increasingly interesting area of research due to its necessity in the biological characterization of the pathways controlling cellular homeostasis and disease development^1,2. Enzymes are responsible for the catalysis of many of the reactions that maintain biological life, controlling necessary pathways such as cellular metabolism^3,4, signaling⁵, and even fundamental processes like genome repair^6,7. Due to their role in these processes, their interactions also play a role in the development of many diseases, as deviations in their activity can cause severe dysregulation in cell activity, causing apoptosis or proliferation of harmful cancer cells². The study of enzymatic activity has had important applications in the development of new therapeutics^8,9, requiring assays that are tailored to each specific enzyme-substrate interaction¹⁰. Multiple enzymatic assays have been established as standard protocols for the evaluation and characterization of these interactions. Assays developed to analyze enzymatic interactions are classified into detection assays that monitor binding for activation/inhibition¹¹ or assays that monitor substrate modification by enzymes¹².

One major role of enzymes is regulating cell behavior. Signal transduction, the intracellular response of a cell to an extracellular trigger¹³, is responsible for cell survival and functionality. Cell proliferation, differentiation, and many other functional processes all involve signaling pathways with enzymatic interactions governing them^14,15. Enzymes catalyze post-translational modifications, which often modulate the massive signaling networks responsible for the correct transmission of extracellular messages¹⁶. Protein phosphorylation is the most common post-translational modification, ubiquitous in cell signaling and multiple other cellular pathways. Consequently, protein kinases have emerged as a significant proportion of potential therapeutic targets due to their critical regulatory role¹⁷. Phosphatases are the natural modulatory molecules for phosphate-based cell signaling complexes^18,19, having the capacity to remove phosphate residues from their target proteins²⁰. In the last decade, phosphatases have become a major therapeutic target for cancer treatment²¹ and inflammatory diseases²² based on their involvement in the regulation of downstream signaling pathways in multiple cell types. Together, protein kinases and phosphatases provide a breadth of interactions, which can be studied through the development of specific enzymatic assay protocols.

Yeast surface display has been used as a tool for the characterization and evaluation of enzymatic activity^23,24. It provides a high-throughput platform for the screening of post-translational modification processes when combined with endoplasmic reticulum sequestration strategies^25,26. This allows kinase-substrate pairs to be co-localized and retained in the endoplasmic reticulum through binding to KDEL receptors²⁷, where phosphorylation of the substrate can occur at increased rates due to the proximity between kinases and their targets. The KDEL receptor binding is mediated by a C-terminal FEHDEL endoplasmic reticulum retention sequence shown to have a stronger retention ability than other HDEL sequences^25,28. The phosphorylated substrate is then anchored to the yeast surface for its subsequent evaluation through flow cytometry²⁹. Currently, there are no generalizable protocols established for the enzymatic modification of substrates displayed on the yeast surface. We expand on the capacities of yeast surface display by taking advantage of the extracellularly expressed phosphorylated substrate variants and modifying them through dephosphorylation by their known phosphatase. Flow cytometry analysis then provides a platform for the phenotypic evaluation of the aforementioned substrates through the measurement of alterations in phosphorylation median as a consequence of the incubation with the known phosphatase. This provides an adaptable method for post-translational modification of surface-displayed proteins while also providing a method for enzymatic modification analysis of interactions when using the yeast surface display platform.

We present techniques for the development and application of an enzymatic modification assay that describes the introduction of a kinase-substrate interaction into the yeast surface display platform, the co-incubation of the expressed phosphorylated substrate with a recombinant phosphatase, and the subsequent analysis of the dephosphorylation activity through flow cytometry. In this report, this is accomplished by co-localizing the cytoplasmic domain of CD28 with lymphocyte kinase (LCK) in the yeast endoplasmic reticulum, followed by display of the phosphorylated CD28 on the yeast surface and subsequent dephosphorylation by Src homology region 2 domain-containing phosphatase-2 (SHP-2). A pan anti-phosphotyrosine antibody (in this study, 4G10), which detects phosphorylated tyrosine residues in a wide variety of peptide sequences, is used for quantification of phosphorylation level as a function of phosphatase treatment. The detailed process provides a generalizable approach for investigating enzyme-substrate interactions; a prospective way of studying enzymes and substrates in purified fashion.

Protocol

1. Cell growth of yeast harboring plasmid and induction of protein expression

1. Following the recipe described in Table 1, prepare the media required for growth of non-plasmid containing yeast (YPD), plasmid-containing yeast cell growth (SD-CAA) and the induction of protein expression (SRG-CAA) as well as SD-CAA plates.
2. Transform yeast display plasmid DNA containing the kinase-substrate pair into EBY-100 yeast cells through the lithium cation-based method^30,31, commonly adopted for use in yeast transformation kits from a variety of manufacturers.
   NOTE: Electroporation³² or other preferred yeast plasmid transformation techniques can be used depending on plasmid construct being transformed.
3. Prepare a 14 mL culture tube with 10 mL of YPD media. Inoculate EBY-100 cells and grow in a shaking incubator at 30 °C, 300 rpm until the culture reaches an optical density (OD_600nm) of 0.8-1.0 (8 x 10⁶- 1 x 10⁷ cells/mL).
   NOTE: OD_600nm is measured by preparing 3 mL sample cuvettes containing 1:10 dilutions of yeast cultures in their respective media and 3 mL blank cuvettes containing the media used for sample dilutions. The OD_600nm program on the spectrophotometer is used to first measure the blank cuvette, then each sample cuvette by setting the corresponding dilution prepared for each sample. 1 OD_600nm corresponds to 1 × 10⁷ yeast/mL.
4. Harvest the cells by centrifuging the culture at 1,000 x g for 3 min, and wash with the 1^st washing solution provided in the yeast transformation kit, or TE (10 mM Tris-HCl and 1.0 mM EDTA)³¹.
5. Pellet the cells again at 1,000 x g for 3 min and resuspend in 1 mL of the transformation buffer provided in the yeast transformation kit, or sterile water. The cells should be aliquoted into 50 µL aliquots and can be stored at -80 °C for up to 6 months.
6. For plasmid transformation, one aliquot per plasmid is prepared and thawed on ice, then, 0.5-1.5 µg of plasmid DNA containing the yeast display construct is added directly to the cells. 0.5 mL of the transformation solution provided in the yeast transformation kit is added, or 0.5 mL of a sterile 50% polyethylene glycol and 0.1 M LiOAc solution³¹. Combine the mixture of cells, plasmid DNA, and transformation solution thoroughly by pipetting.
7. Incubate transformation mixture statically for 30-60 min at 30 °C, vortex mixture at 15 min intervals. Harvest cells by centrifuging at 1,000 × g for 3 min.
8. Prepare a 14 mL culture tube with 4.5 mL of SD-CAA media. Resuspend the cells containing the desired plasmid in 500 µL of SD-CAA and inoculate the prepared 4.5 mL.
9. Being careful not to pierce the agar, distribute 50 µL of the 5 mL of inoculated culture onto a SD-CAA plate and incubate statically at 30 °C for 48 h to determine the transformation efficiency.
10. Incubate the 5 mL of SD-CAA cell culture in a shaking incubator at 30 °C, 300 rpm for at least 18 h. Monitor optical density (OD_600nm) after 16 h and 20 h. Once the sample has grown to a sufficient optical density not exceeding 6, centrifuge the culture for 3 min at 2,500 × g. Discard the supernatant without disturbing the yeast pellet.
11. Resuspend the yeast pellet in SRG-CAA to a final OD_600nm less than 1 (<1 × 10⁷ yeast/mL).
12. Incubate the yeast culture in a shaking incubator at 30 °C, 300 rpm for at least 8 h but no longer than 24 h.
    NOTE: Induction of protein expression in yeast cells can be varied anywhere from 20-37 °C. 30 °C is suitable for synthesis of kinase/substrate pairs^29,33 but can be adjusted if deemed necessary for the specific proteins being studied.
13. Measure OD_600nm to determine the cell density.
    NOTE: The protocol can be stopped at this point by storing the yeast cultures at 4 °C.

2. Biotinylation of 4G10 anti-phosphotyrosine antibody

1. Resuspend a 2 mg vial of PEG4-NHS-Biotin to a final concentration of 5 mM by adding 680 µL of sterile PBS.
   NOTE: Resuspension of PEG4-NHS-Biotin should be done freshly immediately before the biotinylation reaction is carried out. NHS hydrolyzes in aqueous solution. Use of sterile PBS and vials are important for the preparation of reagents for long-term storage and for use in cell-based assays to mitigate any potential effects of contaminants on reagent viability or the sensitive assays being performed.
2. Based on the 4G10 antibody concentration, add 100 µg of antibody into a sterile 1.7 mL vial.
3. Add 1 µL of the prepared 5 mM PEG4-NHS-Biotin from step 2.1 to the vial holding the 100 µg of 4G10 antibody to achieve a molar ratio of biotin to antibody of 7.5:1. Pipette the mixture gently to homogenize the reaction.
4. Incubate the reaction at room temperature with constant rotation for at least 2 h.
5. Follow the manufacturer's protocol for 0.5 mL spin desalting columns to exchange the buffer from the biotinylated 4G10 (B-4G10) into PBS.
   NOTE: Desalting columns with a molecular weight cutoff (MWCO) of 7 kDa are commonly used for biotinylation of antibodies to allow unreacted biotin and other small molecules to be removed while retaining the larger antibody.
6. Dilute the B-4G10 antibody to a final concentration of 1 µM in PBSA (PBS with 1 g/L bovine serum albumin). Aliquot the B-4G10 antibody into smaller volumes to prevent repeated freeze/thaw cycles.
   NOTE: Biotinylated antibodies can be stored at 4 °C for daily usage for up to 3 months without losing significant efficiency. Store the rest of the aliquots not in use at -20 °C for a maximum of 2 years.

3. Dephosphorylation of substrates expressed on the yeast cell surface

1. Prepare the 2x working buffer solution as previously described in the literature³⁴.
   NOTE: 2x is recommended to facilitate the measurement of ingredients needed.
2. Prepare the working buffer for the samples in a 1.7 mL vial by diluting the 2x buffer solution prepared in step 3.1 1:2 in deionized water.
   NOTE: The total reaction volume for each sample will be 20 µL, and each sample will require anywhere between 10 µL and 18 µL of working buffer. Prepare enough working buffer for all samples or controls.
3. Following the recommended sample preparation described in Table 2, label 1.7 mL vials with the corresponding control or sample name.
4. Based on the OD_600nm measured in step 1.9, calculate the volume of culture necessary to recover two million (2 × 10⁶) yeast cells from the corresponding yeast culture for each sample.
5. Add the volume of yeast culture calculated in the previous step into the 1.7 mL vial for each sample.
6. Centrifuge the vial for 1 min at 4,500 × g. Carefully remove supernatant using a micropipette and discard as biohazardous waste.
7. Resuspend the pelleted cells in 1 mL of PBSA and repeat step 3.6.
   NOTE: It is important to remove as much supernatant as possible without disturbing the pelleted cells.
8. Based on its stock concentration, calculate the volume of recombinant human SHP-2 required to have a final concentration of 1,000 nM in a 20 µL total reaction volume.
   NOTE: Recombinant SHP-2 should be aliquoted in small volumes so that any protein used in each assay has not gone through more than two freeze-thaw cycles. Any SHP-2 leftover from an aliquot after all samples have been prepared for an assay should be discarded.
   Stock concentrations of recombinant enzymes can vary depending on lot number. Recombinant SHP-2 is commonly formulated at a stock concentration of 0.2-0.4 mg/mL. For a stock concentration of 0.324 mg/mL SHP-2, this corresponds to a stock concentration of 4.696 µM (SHP-2 has a molecular weight of 69 kDa). 4.26 µL of the SHP-2 stock in a 20 µL reaction results in a final reaction concentration of 1,000 nM SHP-2.
9. Add 7.7 mg of DTT into 10 mL of deionized water prepared in a 15 mL conical to create a 5 mM DTT solution. If equipment limitations make it difficult to weigh milligrams, add 0.77 g of DTT to 10 mL of deionized water, then perform a 100x dilution to create the 5 mM DTT solution used for the assay.
   NOTE: The DTT solution can be prepared in higher concentration stock solutions to be diluted to 5 mM if it is not possible to measure milligram-scale quantities using the equipment available. DTT solution needs to be prepared fresh preceding cell resuspension in working buffer due to its tendency for hydrolyzing, making it unstable over long periods when diluted in water.
10. Resuspend the pelleted cells in the working buffer prepared in step 3.2 so that the final reaction volume in each sample or control is 20 µL.
    NOTE: The amount of working buffer added should be calculated based on the amount of DTT (2 µL) and SHP-2 (calculated in step 3.8) that will be in each sample.
11. Add 2 µL of the 5 mM DTT solution prepared in step 3.9 to each sample or control for a final reaction concentration of 0.5 mM DTT.
12. Add the volume of SHP-2 calculated in step 3.8 to each sample for a final volume of 20 µL and gently mix using a micropipette.
13. Wrap the sample vial lids in parafilm to prevent leakage or cross-contamination.
14. Incubate the samples at 37 °C for 2 h on a rotor at a constant speed.
15. Remove samples from the rotor and stop the reaction by adding 1 mL of PBSA to each sample.
16. Repeat step 3.6.

4. Cell labeling and flow cytometry analysis of dephosphorylated substrates

1. Resuspend samples from step 3.16 in a 20 µL mix of their corresponding primary reagents as described in Table 2. Incubate samples for 20 min at room temperature.
   NOTE: All reagent concentrations used have been calculated to be in excess with regard to the number of proteins expressed on the surface of yeast cells. The calculation assumes the expression of 10,000 proteins/cell from all 2 x 10⁶ yeast in a sample³⁵, when routinely only ~50% of them do. Table 3 shows the excess labeling reagent ratio for each of the reagents expressed in Table 2, calculated as follows:
   
2. Centrifuge samples at 4,500 × g for 1 min and discard the supernatant as biohazardous waste.
3. Wash cells once by resuspending in 1 mL of PBSA. Repeat step 4.2.
4. Resuspend samples in a 20 µL mix of their corresponding secondary reagents as described in Table 2. Incubate samples for 15 min in the absence of light.
5. Repeat step 4.2.
6. Repeat step 4.3.
7. Resuspend the washed samples in 300-500 µL of PBSA and transfer into 5 mL polystyrene tubes to analyze immediately using an appropriate flow cytometer.
   NOTE: If samples need to be transported, keep on wet ice. It is not recommended, but samples can be stored at 4 °C for a maximum of 2 h as wet pellets.
8. After performing necessary startup and preparation of the cytometer for a new experiment, click on the New Experiment button within the File menu, name the experiment, and click Save to ensure data acquired is saved in the desired file path.
9. Select the dot-plot icon within the upper toolbar to create two or more dot-plots for each sample to be run. For one of the dot-plots, select the X-axis name to display the FSC-A channel and the Y-axis name to display the SSC-A channel. This plot shows the Side Scatter - Area versus Forward Scatter - Area and is used to gate yeast cells for further analysis.
10. On another dot-plot, select the X-axis name to display the channel in which the secondary reagent targeting the primary anti-epitope tag antibody fluoresces. Select the Y-axis name to display the channel in which the streptavidin secondary reagent fluoresces. This plot will show only the events gated from the side versus forward scatter plot as yeast cells and is used to display tyrosine phosphorylation on the Y-axis and substrate surface expression on the X-axis.
    NOTE: The secondary reagent targeting the primary anti-epitope tag antibody fluoresces in the FITC (AF-488) channel and the streptavidin secondary reagent fluoresces in the AF-647 channel in this example. Channels used may vary depending on the primary and secondary reagents used during labeling.
11. Place each sample tube in the tube holder of the cytometer and select Run for the cytometer to begin loading the sample and acquiring data. Adjust events to display, events to record, time to record, and sample flow rate as necessary.
12. Define a gate surrounding the healthy yeast cells in the SSC-A vs FSC-A plot created in step 4.9. Figure 1 illustrates a descriptive representation of the gating strategy to apply.
    NOTE: On the SSC-A versus FSC-A plot, 100,000 events to display, and 50,000 events to record within the defined yeast gate is a good guideline to visualize and collect sufficient data for further analysis. More stringent gating strategies to select for singlet yeast can be applied based on a plot of Forward Scatter-Height versus Forward Scatter-Area as recently reported³⁶. The gating strategy shown in Figure 1 corresponds to a less stringent approach, including singlet and some doublet yeast through scatter gating.
13. Record fluorescence of all control samples using a flow cytometry analyzer. Control samples are routinely collected first to help define a gating strategy, as described below in step 4.14.
14. Define a gating strategy for your plot created in step 4.10 prior to analyzing treated samples. Figure 1 illustrates a descriptive representation of the gating strategy to apply.
15. Record fluorescence of dephosphorylated samples using the flow cytometer and the gating strategy defined in steps 4.12 and 4.14.
16. Analyze flow cytometry data acquired using a flow cytometry analysis software.
17. Evaluate dephosphorylation by measuring and comparing the Y-axis median of cells expressing protein on their surface and the baseline phosphorylation provided by non-displaying cells between samples and controls. Calculate the percent median phosphorylation difference as follows:
    

Results

Flow cytometry analysis from an individual replicate of our model system incubated for 2 h without (Figure 1A) and with (Figure 1B) 1,000 nM SHP-2 reveals a median phosphorylation difference of 63.6%, which is defined as the ratio of Y-axis median (phosphorylation) from all surface displayed events minus the baseline phosphorylation signal defined as the Y-axis median of the non-displayed events between the treated sample and the non-treated control as described...

Discussion

The protocol presented allows for the analysis of enzymatic interactions using the extracellular display of proteins on the yeast surface. Incorporating endoplasmic reticulum sequestration into the surface-display plasmid used introduces the capacity to analyze specific interactions between enzymes and post-translationally modified substrates extracellularly due to the intracellular interactions that can be designed to occur^27,29. The previously established enzym...

Materials

Name	Company	Catalog Number	Comments
1 L Media Bottles	Corning	06-414-1D
1.7/2.0 mL Microtubes	Axygen	MCT-175-C
10 µL SureOne Pipet Tips	Fisher Scientific	02-707-438
1000 µL SureOne Pipet Tips	Fisher Scientific	02-707-408
12 mL Polystyrene Round-Bottom Tubes	Greiner	07-000-212
3 mL platic Cuvettes	BRAND	759076D
300 µL SureOne Pipet Tips	Fisher Scientific	02-707-411
5 mL Serological Pipettes	Fisher Scientific	13-678-11D
Acid Casein (Casamino Acids)	Fisher Scientific	BP-1424-500
Analytical Balance	Mettler Toledo	30243397
Bacteriological Petri Dish	Corning	Falcon 351008
Biosafety Cabinets	Labconco	Logic Class II, Type A2  302310102
Biospectrometer	Eppendorf	Kinetic 6136000010
Bovine Serum Albumin	Fisher bioreagents	BP1600-100
Citric Acid	Fisher Scientific	A940-500
CytoFLEX Flow Cytometry Analyzer	Beckam Coulter	Cytoflex C09745	CytExpert software
Dextrose	Fisher Scientific	D16-1
Dithiothreitol	Fisher bioreagents	BP172-5
Donkey anti-goat FITC	Invitrogen	A16000
EDTA	Alfa Aesar	H56165.30
Ez-Link PEG4-NHS-Biotin	Thermo Scientific	A39259
Frozen-EZ Yeast Transformation II Kit	Zymo Research	T2001
Galactose	Fisher Scientific	BP656-500
General Purpose Refrigerator	Marvel Scientific	MS24RAS4RW
Goat anti-myc tag antibody	Bethyl	A190-104A
Mictrotube Centrifuge	Eppendorf	5425 R 5406000313
Mini Low Temperature Refrigerated Incubator	Fisher Scientific	15-015-2632
Mouse anti-phosphotyrosine antibody 4G10	BioXcell	BE0194
Parafilm M	Bemis	M PM999
Phosphate Buffered Saline	Fisher bioreagents	BP399-500
Pipette Controller	Eppendorf	easypet 3 4430000018
Raffinose	Thermo Scientific	J21060-36
Recombinant human Active SHP-2 Protein	R&D Systems	1894-SH
Refrigerated Centrifuge	Eppendorf	5910 R
Saccharomyces cerevisiae yeast surface display strain EBY 100	ATCC	MYA-4941
Shaker Incubator	Eppendorf	M1335-0002 New Brunswick Innova 42
Single Channel Pipette Set	Eppendorf	05-403-151
Sodium Chloride	Fisher Scientific	S671-500
Sodium Citrate Dihydrate	Fisher Scientific	S279-500
Sodium Phosphate Dibasic Heptahydrate	Fisher Scientific	S373-500
Sodium Phosphate Monobasic Monohydrate	Fisher Scientific	S468-500
Streptavidin Alexa Fluor 647	Invitrogen	S32357
Top Loading Balance	Mettler Toledo
Tris hydrochloride	EMD Millipore	648317-100GM
Tube revolver rotator	Fisher Scientific	11-676-341
Weighing Paper	Fisher Scientific	09-898-12B
Yeast Nitrogen Base	BD Difco	291940
Zeba Spin Desalting Columns	Thermo Scientific	89883