Overview¶

Characterize the enzyme kinetics of different enzyme variants involved within the PPK2 energy system family. The goal is to isolate and purify different polyphosphate-based enzymes then evaluate both their independent and encapsulated kinetic activity. Purification will be confirmed via SDS-PAGE. Enzyme activity will be measured via luminescence (ATP/ADP) assays, and protein expression in encapsulated systems via fluorescence and microscopy imaging.

Bill of Materials¶

Protocol¶

Part I – Protein Purification¶

Repeat for each enzyme class.

1. Add a 1g : 2ml ratio of cell pellet to S30 buffer

2. Supplement DTT to your buffer, 2 mM final concentration

3. Sonicate: 5x bursts of 10 sec on, 10 sec off @ 50% amplitude. Record total joules delivered *must keep tube ice cold during sonication

4. Centrifuge at 14,000rpm for 10 minutes @ 4C

5. Collect supernatant and transfer to a new tube, keep on ice

6. Perform spin-column as instructed via provided supplementary material

   • Use Buffer W for wash

   • Use Buffer BXT for elution

Spin Column Procedure – Adapted from iba by Cube Biotech Spin Column Kit Protocol – Version: PR137-0004. Revision date: 10.11.2025¶

• Strep-Tactin®XT 4Flow® high capacity Spin Column Kit

1. Resuspend Strep-Tactin®XT 4Flow® high capacity resin and pipet 100 µL of the 50% suspension into a spin column leading to a column bed volume of 50 µL

2. Centrifuge the sample (maximum speed, 5 min 4ºC) to remove aggregates

3. Apply up to 500 µL sample to the spin column, close the column lid and incubate at room temperature with constant movement (rolling or shaking) for 5-30 min. For most purposes, 5 min are sufficient, but a longer incubation can increase the amount of captured protein, especially for large proteins (≥ 90 kDa)

4. After incubation, open the lid and break off the lower column seal. Place the spin column into a reaction tube and centrifuge for 30 sec at 500 x g.

5. Collect the flow-through for SDS-PAGE analysis and place the spin column into a new reaction tube. Apply 500 µL 1xBuffer W and centrifuge for 30 seconds at 700 x g (Optional: usually, one washing step is sufficient to obtain a highly pure protein, but this step can be repeated if higher purity is required)

6. Collect the washing fraction for SDS-PAGE analysis and place the spin column into a new reaction tube.

   • A. For fast processing, apply 150-200 µL 1x Buffer BXT, close the spin column lid and vortex briefly. After 5 minutes of incubation without movement, vortex again briefly, open the spin column lid and centrifuge for 30 seconds at 700 x g.

   • B. For maximum target protein concentration, apply 50 – 100 µL 1x Buffer BXT, close the spin column lid and vortex briefly. After 5 minutes of incubation without movement, vortex again briefly, open the spin column lid and centrifuge for 30 seconds at 700 x g. Repeat this step one to two times. At least 80% of the target protein will be in the first elution fraction.

Part II – PPK Screen and Standard Curve¶

1. Make a 1 mg/ml BSA solution from your 10 mg/mL BSA stock

   • 40 µL of 10 mg/mL BSA stock with 360 µL of DI water

2. Since stock concentration of PPKs is unknown, run a dilution screen for each protein class to identify which dilution falls within the standard curve. Incubate at room temperature for 10 minutes prior to running

   • See Table 1.

3. Make BSA standard curve solutions following Table 2. Run each standard in duplicate, incubating at room temperature for 10 minutes before running

   • See Table 2.

4. Compare A595 values to your standard curve. Identify the dilution factors whose A595 falls between S1 and S5, ideally near the midpoint.

5. Generate standard curve and calculate concentrations

   • Exclude blank from regression

     1. Plot A595 vs BSA concentration in µg/mL for S1-S5

     2. Fit linear trendline and calculate the concentration of each PPK2 using the trendline equation

        • Multiply by the dilution factor used in step 4 to get the original stock concentration

Table 1. PPK2 dilutions.

Table 2. Bradford dilutions.

Part III – SDS-PAGE¶

1. Add 6uL of your protein sample with 3uL of 4x Laemmli Dye and 4µL of water in a PCR tube

   • DI water is okay and in-fact preferred

2. Add 1uL of your 1M DTT into the PCR tube** (DTT may have already been added)

3. Incubate sample at 95 °C for 5 minutes to denature protein

4. Centrifuge your samples at max speed for 3 minutes.

5. Remove gel from packaging and take off bottom strip and comb on top

6. Place gel into the gel apparatus and ensure that it is sealed properly

7. Rinse wells with DI water first and then dab the wells with a Kim wipe and make sure to get all the water out of the well

8. Pour running buffer in the inner chamber until the buffer covers the wells

9. Fill the outer chamber until you reach the “two gel” mark with running buffer

10. Pre-run the gel at 150 V for about 10 minutes

11. Proceed to load 12uL of your protein sample into your gel including ladder

12. Run your gel at 200V for 40 minutes

13. Remove your gel cassette from the apparatus and image your results

Part IV – Activity Assays¶

ATP Assay¶

Materials:

• PPK2 Reaction Buffer: 50mM Tris-HCl, 10mM MgCl2, 0.5mM DTT, and 0.1mg/mL BSA

• 2x Substrates Mix: 10mM AMP or ADP and 100mM PolyP

• CellTiter-Glo Reagent

• ATP Standard Solutions

• Opaque white 96-well plate

• 1.5mL tubes

Protocols:

Part A: Prepare reagents and reaction tubes

Before starting, thaw all reagents on ice and pre-equilibrate CellTiter-Glo 2.0 to room temperature (protect reagent from light).

1. Label two 1.5 mL microfuge tubes “A” and “B” for the duplicate reactions. Keep on ice.

2. Prepare the PPK2 Enzyme Mix:

   20 µL purified PPK2 enzyme [Enzyme Name:____________Enzyme Class: ______] 180 µL of PPK2 Reaction Buffer.

   Pipet mix gently. Keep on ice.

3. Confirm the 2× Substrate Mix is fully thawed, invert to mix.

4. On ice: prepare 6 microfuge tubes per replicate (A and B) for time-point aliquots by labeling:

   Replicate A: A0, A1, A2, A4, A6, A10

   Replicate B: B0, B1, B2, B4, B6, B10 (A/B = replicates; # = time point in minutes)

5. On ice: prepare 6 microfuge tubes per replicate for 1/10× dilutions by labeling:

   A0d, A1d, A2d, A4d, A6d, A10d

   B0d, B1d, B2d, B4d, B6d, B10d

   ( “d” denotes the diluted aliquot at that time point).

6. To each dilution (“d”) tube, add 18 µL of H2O.

   You will have a total of 24 tubes on ice.

Part B: Assemble the reaction master mix

Assemble the two replicate reactions in tubes A and B. Each reaction master mix is 150 µL total. You will be removing the six time point aliquots from this tube. Read steps 10–13 (Part C) before preparing the reaction master mix You will be collecting time points so its essential that you are ready for next steps before you mix the enzyme with the substrate

Are you ready for steps 10–13?

7. To each of A and B (on ice):

   add 75 µL of PPK2 Enzyme Mix (from part A). Do NOT add the substrate mix yet.

8. Have the 2× Substrate Mix, a timer, and a P200 set to 22 µL ready.

   Plan the order: you will add substrate to reaction A first, then reaction B, staggered ~15 seconds apart so each replicate runs its own timer.

9. Have all 12 time-point tubes (A0–A10, B0–B10) ready on ice

Part C: Start the reaction and collect time points

10. Add 75 µL of 2× Substrate Mix to reaction A, pipet mix three times, start the timer, and IMMEDIATELY pull 22 µL into tube A0 on ice. Repeat for reaction B (~15 s later), collecting into tube B0.

11. Incubate reaction tubes A and B in the 30 °C heat block. Start your timers.

12. At t (min) = 1, 2, 4, 6, and 10 minutes (per replicate clock), remove 22 µL aliquots from the reaction tubes A and B into the corresponding time-point tubes on ice (e.g., A1, A2, ... or B1, B2, ...).

13. After the t = 10 min sample is taken, place the reaction tube back on ice. The reaction is finished.

14. Prepare a 1/10 dilution:

    For each time point aliquot (e.g., A0, A1, A2, ... or B0, B1, B2, ...), transfer 2 µL to the corresponding 1/10 dilution tubes (A0d, A1d, A2d, ... or B0d, B1d, B2d...) containing 18 µL H2O.

15. Prepare ATP standard curve. ATP dilutions have been provided for you.

16. Transfer 20 µL of each ATP dilution to a new microfuge tube, label appropriately

Part D: Add CellTiter-Glo 2.0 and read

17. Using the 8-channel multichannel pipette and a reservoir (if available), dispense 20 µL of CellTiter-Glo 2.0 Reagent into each of the 24 tubes (A & B time points, 1x and 1/10 dilutions).

    Goal: complete reagent addition for all 24 samples within 1–2 minutes.

18. Add 20 uL CellTiter-Glo 2.0 Reagent to each ATP dilution aliquot.

19. Briefly vortex each tube to mix. Cover with foil.

20. Incubate at room temperature in the dark for 10 minutes to equilibrate signal.

Part E: Load the plate

Use rows A through F of a 96-well white opaque plate per group (roughly one third of the plate). Example layout is:

21. Pipette 40 µL from each time-point tube into its assigned well

22. Pipette 40 µL of each ATP standard into the plate

23. Pipette 40 µL of 1× Reaction Buffer into well F1 as a no-ATP blank.

Part F: Analysis

24. Read total luminescence on the GloMax Discover luminometer (Protocol: CellTiter-Glo). Read all wells in a single pass; do not pause between wells.

25. Record raw luminescence values for analysis.

26. Plot raw luminescence (y) vs [ATP] (x) for the standard curve on a log-log plot. Fit a linear region (typically spanning ~3 orders of magnitude).

27. For each time-point well, decide which dilution (1× or 1/10×) falls within the linear range. Use that dilution to back-calculate [ATP] in the original reaction (multiply 1/10× values by 10).

28. Plot [ATP] vs time for each replicate. Compute the mean and standard deviation across the two replicates at each time point.

29. Determine the initial rate (nM ATP / min) from the linear portion of the early time course (typically t = 0 to t = 4 min).

30. Using the protein concentration from Part II (Bradford assay), compute apparent specific activity in nmol ATP / min / mg protein.

31. Report whether the enzyme is active, with the supporting numbers and a qualitative comment on the consistency between the 1× and 1/10× readings.

32. Include all graphs in your notebook.

ADP Assay (Devised by Dr. Oza and Alyssa (Team 2))¶

Part V – Encapsulation¶

PURExpress Encapsulation¶

1. Label three sets of 4 tubes, for each of the following:

   • E—oil emulsion (E1–E4)

   • T—transfer (T1–T4)

   • L—liposomes (L1–L4)

*1 refers to PURExpress only, 2 refers to Class I only, 3 refers to Class II only, and 4 refers to the Class mixture

2. Create the PURExpress cytosol for encapsulation. Keep all reagents and reactions on ice throughout. Preparation on ice ensures that protein expression does not begin during assembly.

   • Pipette the following reagents in the tube labelled E in order from top to bottom using the appropriate pipettes.

• Pipette mix each reaction, then centrifuge the reaction at 5000g [6685 RPM for tabletop centrifuges (r=10 cm)] for 10-30 seconds to spin down bubbles in the reaction.

3. Check the osmolarity of your cytosol using a Vapor Pressure Osmometer before encapsulation.

   • Press “open” on the osmometer and use tweezers to place a circular sample disc on the reading area.

   • Pipette 10μL of your cytosol onto the disc and press “close”.

   • Read and record the osmolarity.

4. Plate reader aliquots. Pipette mix the cytosol solution before transferring 8 uL into two wells of a 384-well plate for kinetics quantification on the Synergy. Set the micropipette to 8 μL and stop at the first stop when dispensing to prevent the introduction of bubbles into the sample.

Encapsulate PURExpress into Liposomes

1. You will be given a range of glucose dilutions labeled with their concentration and osmolarity. Add 300 μL of the correct glucose outer solution, according to the osmolarity of your PURE cytosol, to the tube labeled T.

2. Add 150 μL of the lipid-oil mixture to the top of the PURE reaction in tube E.

3. Emulsify the lipid-oil and PURE mixture by running the tube along a row of empty slots on the 1.5 mL tube rack (watch instructional video for a visual aid on how to do this). Run it down 40–50 times until the solution forms a stable emulsion with an even milky color.

4. Slowly pipette the entire emulsion down the side of the corresponding T tube, so that the emulsion is layered on top of the glucose solution.

5. Centrifuge the T tube at 9000 g [8970 RPM for tabletop centrifuges (r=10 cm)] for 10 min at room temperature to pellet the liposomes.

6. Extract the liposomes from the T tube:

   • Remove the oil layer and lipid debris from the top (supernatant) of the T tube by gently pipetting with a 1000 μL pipette set to 200 μL. Begin at the top of the mixture and aspirate from the solution surface as the liquid level descends in the tube. Dispose of supernatant.

   • Once the majority of supernatant is removed, extract liposomes by pipetting 50 μL of pellet and outer solution from tube T and transfer to the respective liposome tube L. Do not transfer the entire solution. You now have synthetic cells!

7. Hold synthetic cells on ice in the L tube until you are prepared to begin measurement.

8. Pipet 30 µL of each sample into a 384-well glass bottom plate and transfer to microscope.

9. Once data is complete, perform analysis via ImageJ (Fiji) software.

Cytosol Encapsulation¶

Repeat the PURExpress Encapsulation steps above (Part V); however, do not follow the reagent table in step 2a; instead adhere to adjusted conditions below.

Results, Observations, and Conclusions¶

Assays¶

Recorded mass of pellets

FTPPK2 Class I pellet: 0.89g

AJPPK2 Class II pellet 0.9g

Joules delivered through sonicator:

FTPPK2 Class I pellet: 220 J

AJPPK2 Class II pellet 208 J

Purification and SDS-PAGE¶

Conclusion:

As seen in Figure 1, the proteins were pure by SDS-PAGE and EA was used for further steps of the experiment. For both Class I and Class II proteins, each of the proteins fell within range of their expected molecular weight once graphed as seen in Figure 2

Bradford Assay¶

Deviations:

There were originally two BSA Standards for Class I PPK2. However, the data points for the second BSA standard did not fall in the expected curve and were determined that they should be excluded from the data analysis.

Conclusion:

The class I protein dilutions all fell within ~200 µg/mL, while the class II 1:10 and 1:5 dilutions also fell within ~200 µg/mL, while its furthest data point went up to ~650 µg/mL.

PPK2 activity ASSAYS¶

Deviations: In the original protocol/lab manual, it calls for a log-log plot. However, we decided to plot

Table 3. Class I FtPPK2-I ATP, Class II AjPPK2-I ADP, Combination of Class I FtPPK2-I & II AjPPK2-I ATP, and Class III MnPPK2-I ATP time-based assay. Relative luminescence units (RLU) of assay taken on a 96-well white opaque plate on the GloMax Discover luminometer at 30ºC over the course of 10 minutes with assays examined at the 0th, 1st, 2nd, 4th, 6th, and 10th minute. ATP/ADP concentrations calculated of the different proteins and their different dilutions using their RLU with the equation on the graph of Figure 4.

Deviation:

The RLU readings of all the different PPK2 activity assays are out of range of the standard ATP curve.

Conclusion:

The concentrations are not accurate and are solely based off the trendline/equation presented by the ATP standard curve (Figure 4).

Next steps: Make a concentration more dilute than 1/40 as the ATP and ADP assays revealed that the dilutions were not dilute enough as they did not fall within the standard curve.

Deviations: Both the 1:40 and 1:10 dilutions were out of range (much higher than) the ATP Standard curve. The class I & class II did not work as predicted as the data points are scattered throughout the graph. The class III data had a curve different than expected, and this data was collected from another group

Conclusion:

The graph of the activity assay of Class II ADP was included to show that it was active and working as expected showing the upwards-plateauing growth of ADP.

AjPPK2 Class II enzyme showed more activity than FtPPK2 Class I, likely because the ADP assay was completed in a smaller period (1 week) of time compared to the ATP Assay (2 weeks), so the enzyme was able to retain a significant amount of activity compared to the ATP Assay. More specifically, for the ATP assay, the assay was run a week before the luminescence was read, whereas the ADP assay was run a day before the luminescence was read.

Overall, the results were inconclusive as to which energy regeneration system worked the best. The reason being that all of the activity readings were done on separate plates and over the period of a long time causing variation in the experimental environment, causing inconsistencies in relativities throughout the data.

Next steps:

• Incubate Class II with AMP, before combining Class II with Class I. Then, incubate with Class I.

• Testing different combinations of the various Class I and Class II

• Try to do all the experiments in a shorter amount of time so that results are more consistent with each other

• Read all samples and standards on the same plate (the Class III data were read on a separate plate, which prevents direct comparison)

• Have a day to just label centrifuge tubes and prepare dilutions by putting the water into the tubes to maintain same reaction conditions at the very beginning of the experiment, even before purification (very time consuming)

Encapsulation¶

PURExpress Encapsulation¶

Cytosol Encapsulation¶

*Important note, time stamped microscopy was not performed as only initial encapsulation images were obtained. Therefore, microscopic kinetic profiles are not available.

Figure 12. Microscopy screenshot of Class I system at 0 min. The mean green intensity and adjusted intensity values are 227.036 AU and 1.009 AU/µm² respectively.

Figure 13. Microscopy screenshot of Class II system at 0 min. The mean green intensity and adjusted intensity values are 249.732 AU and 0.650 AU/µm² respectively.

Figure 14. Microscopy screenshot of system mixture at 0 min. The mean green intensity and adjusted intensity values are 179.325 AU and 0.448 AU/µm² respectively.

Summary of Results¶

Table 1. Summary of PURExpress results.

Encapsulation Limitations

There were not any significant problems that occurred regarding the encapsulation procedure. Most limitations were presented during data analysis. In terms of the enzyme efficacy, as mentioned earlier and based on assay results, time was a limiting factor, where it seemed that both enzyme classes eventually expire after purification leading to poor results. Therefore, it is critical that encapsulation occurs without delay.

Quantification Limitations¶

Upon analyzing the data, there were inherent errors within the ImageJ (Fiji) software, that is not only exclusive to the user. Some images had issues with microscope focus, which influenced the accurate selection and green intensity measurement of liposomes. Sample size was also a problem, where only one cell was selected and measured, which established generalized conclusions for all other cells. Cell movement must also be considered under the microscope, again limiting accurate measurement. Therefore, conclusions had to consider multiple factors and sets of data.

Conclusions¶

Ultimately, the PURExpress system yielded better results than the cytosol system. Within each respective system, for the PURExpress, the mixture was the most promising, with highest protein translation rates given by the raw mean green and adjusted mean green intensities. The isolated kinetic quantification also confirmed these results with a similar trend. In terms of the cytosol-based system, Class I proved to be most effective, following similar reasoning, but there also is limited data, since microscopic quantification occurred independent of time-dependent fluorescence. Regardless, it appears the mixture was the most effective, supporting initial hypotheses that such a combination could substitute Class III and sufficiently providing exhaustive sources of NMP, NDP, and NTP. Consequently, these cells have extensive supply chains of energy allowing for long lasting protein synthesis and cellular function. Notably, however, inconclusive assay results suggest caution may be necessary before carrying this trend further.