TITLE: Validating TBMs by Fluorescence Polarization (FP)
AUTHORS: Katie Pollock, Michael Ranes, Ian Collins, Sebastian Guettler

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is part of a collection: </span><a href="https://www.protocols.io/private/DA5F6F33163011EBAE360A58A9FEAC2A" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Identifying and Validating Tankyrase Binders and Substrates: A Candidate Approach</span></a></div><br/><div class = "text-block"><span>The poly(ADP-ribose)polymerase (PARP) enzyme tankyrase (TNKS/ARTD5, TNKS2/ARTD6) uses its ankyrin repeat clusters (ARCs) to recognize degenerate peptide motifs in a wide range of proteins, thereby recruiting such proteins and their complexes for scaffolding and/or poly(ADP-ribosyl)ation. Here, we provide guidance for predicting putative tankyrase-binding motifs, based on the previously delineated peptide sequence rules and existing structural information. We present a general method for the expression and purification of tankyrase ARCs from</span><span style = "font-style:italic;">Escherichia coli</span><span>and outline a fluorescence polarization assay to quantitatively assess direct ARC–TBM peptide interactions. We provide a basic protocol for evaluating binding and poly(ADP-ribosyl)ation of full-length candidate interacting proteins by full-length tankyrase in mammalian cells.</span></div></div>

[STEPS]
?. [3.2.2 Purification of Tankyrase ARCs]
A similar procedure is followed for the purification of all tankyrase ARCs. All buffers are ice-cold, and work is performed at 4 °C or on ice throughout. In the first step, the protein is affinity-purified by immobilized metal affinity chromatography (IMAC) using a Ni2+ affinity column. The affinity tag is then cleaved overnight using TEV protease, under simultaneous dialysis to remove imidazole from the elution step. A second Ni2+ affinity chromatography step separates the cleaved tag from the protein. An optional anion exchange chromatography step removes further impurities. Finally, size exclusion chromatography yields high-quality purified protein for downstream applications. The general procedure is applicable to all TNKS and TNKS2 ARCs with small changes (Fig.2a).For ARC2-3 and ARC1-5 constructs described here, a minimal NaCl concentration of 300 mM needs to be maintained, compared to 100 mM for all single-ARC constructs, and glycerol may further help stabilize the protein [7].
?. [3.2.3 Fluorescence Polarization Assay for TBM Validation]
This assay measures the direct binding of a fluorescently labeled TBM candidate peptide to a tankyrase ARC [7]. The ARC protein is titrated at a constant peptide probe concentration, and the change in polarization (ΔFP) is measured and plotted. Plotting ΔFP values against [ARC] enables a dissociation constant (Kd) to be calculated. In the present example, we use fluorescein-based peptide labels, but the general method can be adapted for different fluorophores.
?. [Expression of Tankyrase ARCs]
Transform chemically competent BL21-CodonPlus (DE3)-RIL E. coli cells with tankyrase ARC expression constructs by heat shock. Plate cells on LB agar plates containing suitable antibiotics, here  and . Incubate at .
[kanamycin]
[chloramphenicol ]
37 °C
We recommend using vectors with a kanamycin selection marker. Ampicillin hydrolysis by secreted β-lactamase and under low pH increases the proportion of cells lacking the plasmid, which decreases protein yield [33].
RIL cells contain additional tRNAs for codons of Arg, Ile, and Leu that are otherwise rare in E. coli. The plasmid bearing these genes contains a chloramphenicol selection marker.
?. [Expression of Tankyrase ARCs]
Pick a single colony from the plate and inoculate an overnight pre-culture of  supplemented with  and . Incubate  in a shaking incubator at  .
[LB medium]
[kanamycin]
[chloramphenicol ]
37 °C
?. [Expression of Tankyrase ARCs]
Add  to each liter of TB medium supplemented with  and . Generally, the yield for individual TNKS and TNKS2 ARC constructs is 6–10 mg/L of culture.
[pre-culture]
[kanamycin]
[chloramphenicol ]
?. [Expression of Tankyrase ARCs]
Incubate cultures at  with shaking at  until an optical density at 600 nm (OD600) of approximately 2.0 is reached. Remove cultures from incubator and cool them at  for .
37 °C
33
4 °C
Compared to LB, TB is richer and enables higher cell densities in the log phase of growth. Cooling the cultures before IPTG induction slows down expression, thereby facilitating correct protein folding and increasing protein solubility.
?. [Expression of Tankyrase ARCs]
Add IPTG to a final concentration of  (0.5 mL of 1 M stock per liter of culture) to induce protein production. Incubate cultures at  with shaking (≈15 h).
18 °C
?. [Expression of Tankyrase ARCs]
Collect cells by centrifugation in a precooled centrifuge at .
Centrifuge: 4000 34, 30 min, 4 10
?. [Expression of Tankyrase ARCs]
Discard supernatant. The cells can be lysed for immediate purification. Alternatively, pellets can be flash-frozen in a liquid nitrogen bath for storage at  until required .
-80 °C
?. [3.2.2 Purification of Tankyrase ARCs]
Dissolve protease inhibitor tablets in the required volume of lysis buffer (approximately  for every ) and add lysozyme ( final) in a large glass beaker containing a magnetic stirrer bar. Add frozen cell pellet and stir gently until cells are completely thawed and a homogeneous suspension is obtained.
[buffer]
[cell pellet]
?. [3.2.2 Purification of Tankyrase ARCs]
Break cells by using a large-tip sonicator at 40% amplitude output, using a on,  off pulse, for a total of . Keep cells  continuously to avoid overheating. Take a sample of the total lysate for subsequent analysis by SDS-PAGE ( + ).
[ “on” time]
on ice
[lysate]
[4× SDS sample buffer]
For large volumes, it may be easier to lyse cells in two batches. Alternative disruption techniques can be used, such as homogenization by an Avestin EmulsiFlex homogenizer.
?. [3.2.2 Purification of Tankyrase ARCs]
Remove cell debris and insoluble material by centrifugation at . If lysate is still cloudy, centrifuge for a further  until clear.
Centrifuge: 30000 34, 30 min
?. [3.2.2 Purification of Tankyrase ARCs]
While the lysate is being cleared, prepare a  by washing with  and then equilibrating with  for Ni2+ affinity column, using a low-pressure peristaltic pump, set to a flow rate of 2 mL/min.
[HisTrap Ni2+ affinity column]
[H2O]
[buffer A]
We recommend using one 5 mL column per 4 L expression culture. If using larger volumes, connect additional column(s) in series. Ni2+ affinity columns can easily be reconstituted by stripping from and re-charging with Ni2+ as per the manufacturer’s instructions.
?. [3.2.2 Purification of Tankyrase ARCs]
Decant the supernatant into a beaker, and sonicate ,  on,  off for a total of   to further break up genomic DNA and avoid clogging the filter in the next step.
on ice
[“on” time]
?. [3.2.2 Purification of Tankyrase ARCs]
Filter the supernatant through 5.0 μm syringe filter units. Take a sample of the soluble lysate for subsequent analysis by SDS-PAGE ( + ). Load the cleared lysate on the equilibrated HisTrap column using the peristaltic pump as before. The His6-GST-tagged protein will bind to the immobilized Ni2+ ions. Wash the column with  to remove unbound contaminants. Collect lysate flow-through and the wash buffer in case the protein was not bound or was released during the wash, and take samples for analysis by SDS-PAGE ( + ).
[lysate]
[4× SDS sample buffer]
If available with the FPLC setup, a superloop or, preferably, a sample pump can be used to load the column.
[buffer A (100 mL)]
[flow-through or wash]
[4× SDS sample buffer]
?. [3.2.2 Purification of Tankyrase ARCs]
Attach the HisTrap column to an FPLC system, and elute the His6-GST-ARC protein with the following three-step protocol, at a flow rate of 2 mL/min, collecting 1.5 mL fractions in a 96-deep-well block:
?. [3.2.2 Purification of Tankyrase ARCs]
Take samples of each peak elution fraction ( +  ). Analyze the samples taken in steps 11 and 15 (2 μL of sample for lysates and flow-through,  for wash) and the peak elution fractions () by SDS-PAGE (Fig. 2b).
[eluate]
[4× SDS sample buffer]
[of sample]
[of sample]
?. [3.2.2 Purification of Tankyrase ARCs]
Pool fractions containing ARC fusion protein. Take a sample of the pooled fractions ( + ). Add TEV protease (approximately   for every ) to cleave the tag.
[protein]
[4× SDS sample buffer]
[of 5 mg/mL stock]
[fusion protein solution]
?. [3.2.2 Purification of Tankyrase ARCs]
Soak dialysis tubing (3500 Da MWCO) in dialysis buffer, and seal one end with a dialysis membrane clip. Transfer the protein into the dialysis tubing and seal the other end. More than one dialysis tube may be required. Dialyze  in a 2 L glass beaker containing dialysis buffer, with gentle stirring at . Avoid contact between the stirrer bar and the dialysis tubing to prevent rupture.
4 °C
?. [3.2.2 Purification of Tankyrase ARCs]
Pour content of dialysis tubing into 50 mL Falcon tubes. Centrifuge at  to remove any precipitate that may have formed. Take a sample of the protein post-TEV cleavage ( + ). White precipitate often forms overnight with the change to low-salt buffer. SDS-PAGE analysis of the precipitate reveals it to be enriched in a contaminant (not shown).
Centrifuge: 3200 34, 15 min
[protein]
[4× SDS sample buffer]
?. [3.2.2 Purification of Tankyrase ARCs]
Meanwhile, wash a  with . Next, equilibrate the column in . Filter the supernatant through a 0.22 μm syringe filter and load it onto the equilibrated HisTrap column using the low-pressure peristaltic pump. Collect the flow-through. The untagged ARC protein will pass through, while the cleaved tag (or residual His6-GST-ARC fusion protein) will be retained on the column.Wash with 2 CV of dialysis buffer to fully remove all ARC protein from the column, collecting the wash. Take samples of the flow-through and wash ( + ).
[HisTrap column]
[H2O]
[dialysis buffer]
Depending on the ARC construct and the final concentration of imidazole in the dialysis buffer, ARCs can bind weakly to the Ni2+ column even after tag cleavage . They can be eluted with a further imidazole gradient. While this adds one more step to the purification protocol, it enables even higher purities to be achieved.
[protein]
[4× SDS sample buffer]
?. [3.2.2 Purification of Tankyrase ARCs]
Analyze , post-TEV sample and flow-through/wash samples from the second Ni2+ column by SDS-PAGE to assess successful tag removal and separation (Fig. 2c).
[pre-TEV sample]
?. [3.2.2 Purification of Tankyrase ARCs]
Take the protein-containing samples (flow-through/wash) and concentrate the protein to about  by centrifuging in a 3000 Da MWCO spin concentrator at  at a time in a swinging bucket rotor. Mix by gently pipetting up and down to prevent a steep concentration gradient from forming in the concentrator. Take a sample of the concentrated protein ( + ).
20 mL
Centrifuge: 3200 34, 30 min
[protein]
[4× SDS sample buffer]
?. [3.2.2 Purification of Tankyrase ARCs]
The following anion exchange chromatography step is optional, but was found to increase the purity of ARC proteins, especially TNKS2 ARC5, by removing a higher-molecular-weight impurity:Using the peristaltic pump at 2 mL/min , wash a 5 mL HiTrap Q HP column with , followed by  for Q column, and then equilibrate with  for Q column. Pass the concentrated protein over the column, and collect the flow-through. Wash the column with  for Q column, also collecting the flow-through. ARCs do not bind to the Q column; however, the impurity does and will be retained on the column. Take a sample of the combined flow-through and wash (+ ). Analyze  from step 23 and this step by SDS-PAGE as above (Fig. 2d).
[H2O]
[buffer B]
[buffer A]
[buffer A]
The Q column step can also be performed before concentration, directly using the flow-through from the second Ni2+ affinity column; however, prior concentration saves time in loading the column.
[protein ]
[4× SDS sample buffer]
[samples]
?. [3.2.2 Purification of Tankyrase ARCs]
Concentrate the protein as in step 23, to ≤: Take the protein-containing samples (flow-through/wash) and concentrate the protein by centrifuging in a 3000 Da MWCO spin concentrator at  at a time in a swinging bucket rotor. Mix by gently pipetting up and down to prevent a steep concentration gradient from forming in the concentrator. Take a sample of the concentrated protein ( + ).
2 mL
Centrifuge: 3200 34, 30 min
[protein]
[4× SDS sample buffer]
?. [3.2.2 Purification of Tankyrase ARCs]
Centrifuge protein in a microcentrifuge tube at  to remove any particles before size exclusion chromatography.
Centrifuge: 18000 34, 10 min
?. [3.2.2 Purification of Tankyrase ARCs]
Load the concentrated protein onto an equilibrated size exclusion column connected to an FPLC system , using a 2 mL sample loop.
Use a Superdex 200 column for ARC1-5 constructs. All other ARC constructs are sufficiently small for the Superdex 75 column.
[size exclusion buffer]
?. [3.2.2 Purification of Tankyrase ARCs]
Take samples of the input material and peak fractions ( or  + ). Analyze  by SDS-PAGE (Fig. 2e).
[input]
[fraction]
[4× SDS sample buffer]
[samples]
?. [3.2.2 Purification of Tankyrase ARCs]
Combine fractions containing pure protein. Wash the membrane of a spin concentrator by spinning through one concentrator volume of H2O to remove any stabilizing agents such as glycerol and azide. Now concentrate the protein using the pre-washed spin concentrator as described above (step 24) until a concentration of > is achieved (solubility of multi- ARC constructs can be lower). The concentration can be determined by measuring the UV absorbance at 280 nm and using the molar extinction coefficient calculated by ProtParam (http://web.expasy.org/protparam/) [29].
?. [3.2.2 Purification of Tankyrase ARCs]
Aliquot the protein into small volumes in thin-walled PCR tubes and flash-freeze them in a liquid nitrogen bath. Frozen protein aliquots can be stored at  until future use.
-80 °C
?. [3.2.2 Purification of Tankyrase ARCs]
step 1: .
[100% buffer A]
?. [3.2.2 Purification of Tankyrase ARCs]
step 2: over .
[gradient from 0–100% buffer B]
?. [3.2.2 Purification of Tankyrase ARCs]
step 3: .
[100% buffer B]
?. [3.2.3 Fluorescence Polarization Assay for TBM Validation]
Ensure the plate reader is set up for fluorescence polarization, with the correct filters for the fluorophore . The fluorescein example uses a 485 nm excitation filter and two 520 nm emission filters, one parallel and one perpendicular to the plane of the linearly polarized light used for excitation.
?. [3.2.3 Fluorescence Polarization Assay for TBM Validation]
Dissolve the lyophilized peptide probe in FP assay buffer, to approximately . The concentration can be measured spectrophotometrically. Here, we measure the absorbance of the peptide stock at 492 nm and use a molar extinction coefficient for fluorescein of ε492 = 83,000/M/cm [30]. The concentrated probe can then be aliquoted into small volumes (5–10 μL) and flash-frozen in liquid nitrogen for long-term storage at , to be thawed when required.
Confirm the pH of the peptide stock solution before measuring the concentration. Acidity, for example due to residual trifluoroacetic acid from the peptide synthesis, will strongly affect fluorophore absorption.
-80 °C
?. [3.2.3 Fluorescence Polarization Assay for TBM Validation]
Determine the optimal probe concentration to use. This will depend on the sensitivity of the plate reader. FP values should be independent of the probe concentration used, and give a robust signal window for an unbound vs. protein-bound peptide. Make 2× stocks of a twofold dilution series of probe in FP buffer, from 0–800 nM (10 concentrations + no peptide). It is often easier to make a 1/100 dilution of the concentrated peptide stock to avoid pipetting small volumes. Total sample volumes per well are either 15 or 50 μL (see step 36 below).
?. [3.2.3 Fluorescence Polarization Assay for TBM Validation]
Prepare a 2× protein stock solution, by diluting tankyrase ARC protein in FP buffer to  (10 μM final protein concentration). The protein concentration should be in vast excess over the peptide probe to obtain a first impression of the anticipated signal window between free and protein-bound peptide probe .
?. [3.2.3 Fluorescence Polarization Assay for TBM Validation]
Plate out the assay samples in technical duplicate (see Table 2 and note below). Small-volume plates will require 15 μL per well ( of each stock), standard-volume plates  per well ( of each stock). Mix the probe stock solution 1:1 with either FP buffer for the peptide probe-only titration or protein stock for the titration in the presence of protein. Add a “blank” well with FP buffer only. A well with  (literature FP of 35 millipolarization units, mP) can be used as a standard to automatically set the gain adjustment of the plate reader. Temporarily seal the plate with adhesive film or a lid and spin at  in a swinging bucket rotor with plate inserts. Incubate in the dark at  for  to ensure binding equilibrium is reached.   ABCDEFGHIJKLMNO112345678910111213142A3B00.781.563.136.2512.52550100200400BlankStandard4C00.781.563.136.2512.52550100200400BlankStandard5D00.781.563.136.2512.52550100200400BlankStandard6E00.781.563.136.2512.52550100200400BlankStandard
7.5 µl
50 µl
25 µl
[fluorescein]
Centrifuge: 1000 34, 1 min
80 Room temperature
ABCDEFGHIJKLMNO112345678910111213142A3B00.781.563.136.2512.52550100200400BlankStandard4C00.781.563.136.2512.52550100200400BlankStandard5D00.781.563.136.2512.52550100200400BlankStandard6E00.781.563.136.2512.52550100200400BlankStandard
We recommend leaving wells in the outermost rows and columns empty to reduce the microplate “edge effect,” a discrepancy in readings between the central and peripheral wells [36].
?. [3.2.3 Fluorescence Polarization Assay for TBM Validation]
Set the gain adjustment of the instrument. The POLARstar Omega automatically calculates the gain by scaling the FP value to 35 mP for the wells containing . 50 flashes per well is a good starting point to minimize fluorophore bleaching, and means each data point will be the average of 50 measurements.
[free fluorescein]
?. [3.2.3 Fluorescence Polarization Assay for TBM Validation]
Read the plate and calculate FP in mP. Some plate readers and software packages will automatically blank-correct and calculate the FP values. If not, subtract the averaged F_parallel and F_perpendicular readings obtained for the blank wells from the corresponding individual values of the other wells. FP values are calculated using the following equation:FP (mP)=\frac{(F\_parallel−F\_perpendicular)}{(F\_parallel+F\_perpendicular)}F_parallel and F_perpendicular denote the fluorescence intensities parallel and perpendicular to the plane of the linearly polarized lightused for excitation. mP, millipolarization units.
?. [3.2.3 Fluorescence Polarization Assay for TBM Validation]
Export the FP data and raw fluorescence intensity values. Plot the data (average FP vs. [probe]) using GraphPad Prism or your data analysis program of choice (Fig.3a).
?. [3.2.3 Fluorescence Polarization Assay for TBM Validation]
Determine the minimum probe concentration required. Raw fluorescence intensity values of >10× the blank reading are necessary to calculate accurate FP values. In the example shown in Fig. 3a, fluorescence intensity values measured with probe concentrations below  were too low and therefore excluded.
?. [3.2.3 Fluorescence Polarization Assay for TBM Validation]
Determine the maximum probe concentration. The FP values will drop sharply when the signal overloads the detectors. In this case, both detectors reach saturation, so the fluorescence intensities registered in the parallel and perpendicular channels will be identical, and hence the FP calculated will be zero. In the example shown in Fig. 3a,  is the maximum usable peptide probe concentration. FP readings between 6.25 and 50 nM probe are approximately constant.
?. [3.2.3 Fluorescence Polarization Assay for TBM Validation]
Peptide probe concentrations should be kept as low as reasonably possible. Thus, the peptide concentration can be ignored in Kd calculations. 25 nM probe was chosen for the protein titration experiments shown below. Prepare a 2× probe stock solution ( in FP buffer). Prepare a twofold dilution series of tankyrase ARC protein from 0–400 μM (0–200 μM final concentration) in FP buffer. The maximum protein concentration may be lowered if Bmax is reached at low protein concentrations, i.e., in the case of high affinity. An initial maximum [ARC] of  ensures that binding can be detected for weak binders as well.
?. [3.2.3 Fluorescence Polarization Assay for TBM Validation]
Set up a binding experiment for Kd determination. Mix the probe and protein stock solution 1:1 to give  (7.5 μL of each stock solution, small-volume plates). Centrifuge the plate as in step 39 (spin at  in a swinging bucket rotor with plate inserts) above. Incubate the plate in the dark at  for  to achieve equilibrium before measurement.
[final well volume]
Centrifuge: 1000 34, 1 min
80 Room temperature
?. [3.2.3 Fluorescence Polarization Assay for TBM Validation]
Calculate FP as in step 38 above and export the raw fluorescence and calculated FP values.
?. [3.2.3 Fluorescence Polarization Assay for TBM Validation]
Enter the individual FP values into GraphPad Prism or your data analysis program of choice.
?. [3.2.3 Fluorescence Polarization Assay for TBM Validation]
Baseline-correct by subtracting the FP value for the zero-protein well from all other values in that row, to obtain ΔFP values. Calculate the mean of the technical duplicates. Plot ΔFP against [protein]. Perform a nonlinear regression analysis, using a one-site total binding model:ΔFP = Bmax × [ARC]/(Kd + [ARC]) + NS × [ARC] + BackgroundBmax denotes the maximum specific binding in mP, where the binding curve saturates; K d is the dissociation constant ; NS denotes the slope of nonspecific binding in ΔFP/[ARC] units; Background denotes the baseline ΔFP value, which should be zero. Although it is possible to constrain the background to zero, it is advisable to retain the background term since the baseline is defined by more values than that from the zero-protein sample.Non-binding peptides will give a straight line of nonspecific binding. Binding peptides will give a logarithmic curve (Fig. 3b). This analysis will yield a Kd value.
?. [3.2.3 Fluorescence Polarization Assay for TBM Validation]
Repeat the experiment to obtain pooled data from (typically three) separate experiments . In each separate experiment, technical duplicates are combined into a single data point. Error bars (SEM) are calculated for the mean data points from the separate experiments (Fig. 3b). The peptides from 3BP2 , TRF1 , and MERIT40 (two peptides) bind TNKS2 ARC5 with the indicated affinities, while no binding is detectable for the G6R negative control peptide from 3BP2 (Fig. 3b).
?. [Expression of Tankyrase ARCs]
cDNA sequences encoding TNKS or TNKS2 ARC constructs are cloned into a pETM-30-2 expression vector (or similar vector) to generate fusion proteins with an N-terminal His6-GST tag followed by a TEV protease cleavage site (ENLYFQG) [7]. See Table 1 for suggested ARC boundaries [7]. Chemically competent BL21-CodonPlus (DE3)-RIL E. coli cells for protein expression are transformed with sequence-validated plasmids. A single colony is selected to inoculate an overnight pre-culture, which is then used to inoculate the larger expression cultures (typically 4–8 L). The cultures are grown shaking at 37 °C to exponential phase, then cooled at 4 °C for 30 min before induction with IPTG overnight at 18 °C. The cells are then collected by centrifugation. Purification can either follow immediately, or cell pellets can be stored at −80 °C until processed for purification.