Protocol for purification and enzymatic characterization of members of the human macrophage migration inhibitory factor superfamily

Summary

Macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT or MIF-2) are two proteins serving a key role in the pathogenesis of multiple disorders, including cancer.¹ Here, we present a protocol for the purification and enzymatic characterization of MIF and D-DT using keto-enol tautomerase activity. This approach measures enzymatic activity through the formation of an enol-borate complex. We describe steps for expressing and purifying proteins, preparing the 96-well microplate, and assay implementation including monitoring of keto-enol tautomerase activity.

For complete details on the use and execution of this protocol, please refer to Parkins et al.^2,3

Graphical abstract

Highlights

• •Systematic protocol for the purification and enzymatic characterization of MIF/D-DT
• •Describes common pitfalls and potential solutions
• •Includes user-friendly procedures

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Macrophage migration inhibitory factor (MIF) and D-dopachrome tautomerase (D-DT or MIF-2) are two proteins serving a key role in the pathogenesis of multiple disorders, including cancer.¹ Here, we present a protocol for the purification and enzymatic characterization of MIF and D-DT using keto-enol tautomerase activity. This approach measures enzymatic activity through the formation of an enol-borate complex. We describe steps for expressing and purifying proteins, preparing the 96-well microplate, and assay implementation including monitoring of keto-enol tautomerase activity.

Before you begin

Besides the keto-enol tautomerization assay, this protocol also contains key information on the production of active MIF and D-DT. Further support on cloning, recombinant protein overexpression and purification can be found here.⁴ The keto-enol tautomerization assay can be used to compare catalytic activities of MIF and D-DT variants (e.g., mutations) as well as to determine the potency of small molecule modulators.

Protein expression

Timing: 4 days

• 1.
  Transform BL21 (DE3) competent cells with the plasmid of interest (human wild-type (WT) MIF and human WT D-DT are encoded in pET-11b and pET-22b, respectively).

  Note: If more plasmid is needed, one can use a similar transformation protocol but instead of BL21 (DE3), use XL-10 competent cells.

  Note: The respective MIF and D-DT plasmids have been well documented to support protein expression in high quantities. Nevertheless, MIF and D-DT may also be expressed by other plasmids.

  Note: The steps below are for heat-shock transformation; however, other transformation methods may also be used.

  • a.
    Make Luria Broth (LB) agar plates containing 100 μg/mL ampicillin and store them at 4°C until ready for use. Per plate, pour ∼25 mL of warm LB agar solution and avoid bubbles.
  • b.
    Thaw out the competent cells on ice.

    Note: The competent cells used were purchased, not made in house. The procedure may have to be optimized for homemade competent cells.

  • c.
    Add 500–700 ng of plasmid to your competent cells (50 μL).
  • d.
    Let the mixture incubate on ice for 10 min.
  • e.
    Heat shock at 42°C for 30 s.
  • f.
    Put the cells back on ice to recover for 2 min.
  • g.
    Dilute the cells up to 1mL with sterile LB.

    Note: Only for this step, the LB media should be antibiotic-free.

  • h.
    Incubate the cells at 37°C while shaking for 45 min.
  • i.
    Spin down the cells at 4°C using a tabletop centrifuge’s pulse option up to max speed.
  • j.
    Remove 800 μL of the supernatant.
  • k.
    Resuspend the pellet in the remaining 200 μL.
  • l.
    Plate the cells in an LB agar plate containing 100 μg/mL ampicillin.

    Note: multiple plates (1–3) with varying cell concentration may be used at this step to ensure that single cell colonies will be obtained.

  • m.
    Incubate the two plates for 12–16 h.
• 2.
  Creating a glycerol stock.

  • a.
    Select a single colony from one of the plates with a sterile pipette tip.

    Note: Selecting the colonies with a 200–1000 μL tip will help mix and aerate the media/cells in later steps.

  • b.
    Inoculate a culture tube containing 2 mL of LB-ampicillin (100 μg/mL).
  • c.
    Incubate at 37°C, under shaking (200 rounds per minute (RPM)) for 12–16 h.
  • d.
    Transfer the 200 μL of 100% glycerol to a freezer tube.
  • e.
    Add 800 μL of the culture.
  • f.
    Vortex to mix.
  • g.
    Store at −80°C.

    Pause point: The glycerol stock can be kept in a -80°C freezer and should only be removed in an insulated bag containing dry ice.

• 3.
  Protein expression.

  Note: This procedure is for 1L worth of cells but can be proportionally adapted to any cell volumes.

  • a.
    Create a started culture.

    • i
      .Remove the glycerol stock from the −80°C freezer and place the vial on dry ice.
    • ii
      .Scrape the surface of glycerol stock with a sterile tip (200–1000 μL tip).
    • iii
      .Inoculate a flask containing LB /100 μg/mL ampicillin.

      Note: The volume of started culture should be 1/100^th of the final culture (for an example, see step 3b).

    • iv
      .Incubate at 37°C, under shaking (200 RPM), for 12–16 h.
    • v
      .Return the glycerol stock back to the −80°C freezer.

      Note: The glycerol stock can be reused many times. Use a cold shuttle or dry ice to remove it from the -80C and do not let the glycerol stock thaw.

  • b.
    Grow the cells to an optical density at 600 nm (OD₆₀₀) equal to 0.6 to 0.8.

    • i
      .Inoculate 1 L of LB supplemented with 100 μg/mL ampicillin with 10 mL of started culture.

      Note: the total volume of your cell culture should not exceed 25% of the flask’s volume capacity.

    • ii
      .Incubate at 37°C while shaking until the OD₆₀₀ reaches 0.6–0.8 (∼2 h 30 min).
    • iii
      .Induce protein expression.
    • iv
      .Add Isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM.
    • v
      .Incubate while shaking at 37°C for 4 h.
  • c.
    Collect and store cells.

    • i
      .Spin down cells at 4 °C at 4120 × g for 12 min.

      Note: For this step, it is recommended to use centrifugation bottles that can hold a volume > 250 mL in order to collect the cells in a gentle, yet timely manner.

    • ii
      .Resuspend cells with 40 mL (per liter of expression culture) of lysis buffer and transfer to a 50 mL centrifuge tube.
    • iii
      .Pellet cells at 4 °C at 25,150 × g for 12 min.
    • iv
      .Remove supernatant.
    • v
      .Store cells at −80°C.

      Pause point: The pelleted cells can be stored in a −80°C freezer until protein purification.

Protein purification

Timing: 8 h

• 4.
  Cell Lysis.

  • a.
    Thaw the pellet at 22°C.
  • b.
    Resuspend the cells in 20 mL of lysis buffer (20 mL lysis buffer/1L expression culture.

    Note: MIF lysis buffer - 20 mM Tris.HCl, 20mM NaCl, pH 7.40.

    Note: D-DT lysis buffer - 20 mM Tris.HCl, 20mM NaCl, pH 8.50.

  • c.
    Put the resuspended cells into a container with ice.

    Note: This step is to keep the cells cold during the lysing process.

  • d.
    Sonicate at 60% power for 10 s on/50 s off for a total time of 24 min.

    Note: Time and power may vary between sonicators. The sonication parameters have to be optimized based on the sonicator under use. Non-optimal sonication parameters may cause protein aggregation or incomplete cell lysis.

  • e.
    Spin down the cells for 50 min at 25,150 × g at 4°C.
  • f.
    Collect the supernatant (cell lysate).
  • g.
    Filter the cell lysate with a 0.22 μm PES syringe filter.
• 5.
  Purify the protein via anion exchange chromatography (Q-Sepharose column).

  Note: For protein purification a fast protein liquid chromatography (FPLC) system is required.

  • a.
    Equilibrate a Q-Sepharose column (75–120 mL column for MIF or 5 mL column for D-DT) with the proper lysis buffer for each protein (see protein purification section, cell lysis step).
  • b.
    Load the filtered cell lysate onto the column at 1 mL/min flow rate.

    Note: One can begin collecting fractions at this point in order to ensure that no protein is lost in the flow through due to mistakes during buffer making, equilibration, and/or loading processes.

  • c.
    After loading, continue the run using lysis buffer at 2 mL/min.
  • d.
    MIF will come off without binding to the column, D-DT will need to be eluted off the column with 5% elution buffer (20 mM Tris.HCl, 1 M NaCl, pH 8.50).

    Note: Purity of the protein may vary based on the length of the column, the type of Q-Sepharose beads, and how clean/well maintained the column is. Smaller, higher resolution columns can be used in series with a larger column to provide a purer product, if necessary. In this protocol, the larger MIF column contained Q Sepharose XL beads meanwhile the 5 mL column used for the D-DT purification was a HiTrap Q HP pre-packed column.

  • e.
    Analyze the purity of the protein with a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel; in both cases (MIF and D-DT), an overexpressed protein band should be clearly visible near 12 kDa. After the Q-Sepharose step, MIF will be ≥ 95% pure while D-DT will come off the column at lower purity.

    Note: The elution fractions can be collected and stored for 12–16 h at 4°C, however, to ensure a well-behaved protein, it is advised to move on to size-exclusion chromatography as soon as possible. This is critical especially for D-DT.

• 6.
  Purify the protein by size-exclusion chromatography (SEC).

  • a.
    Equilibrate a S75 16/600 SEC column with the lysis buffer of the desired protein.
  • b.
    Filter the protein fractions obtained from the Q-Sepharose purification step with a 0.22 μM PES syringe filter.

    CRITICAL: Filtering the protein before concentration removes any existing precipitate and helps reduce the chance of precipitate forming during concentration.

  • c.
    Concentrate the protein down to 3 mL (or 3/5^th of the total loop volume) using a 15 mL Amicon concentrator with a 10 kDa molecular weight (MW) cut off. Centrifuge at 2100 × g at 4°C for 10–12 min at a time, mixing in between spins.

    CRITICAL: While mixing, check for precipitate and if any has formed, filter the sample immediately.

  • d.
    Filter the concentrated sample with a 0.22 μM PES syringe filter.

    Note: For >1 mL loading volumes, transfer the sample to a 1.5 mL microcentrifuge tube and centrifuge using a tabletop centrifuge. The sample should be centrifuged at max speed for 5 min, at 4°C. Check for a pellet and remove the supernatant. If needed sacrifice a few μL of sample to ensure that potential precipitation is not transferred into the supernatant.

    CRITICAL: Filtering after concentration will remove any non-visible precipitate, which may damage the SEC column. If large amounts of precipitate formed during concentration, it would be recommended to restart the purification from step 4a.

  • e.
    Load the sample onto the column through a loop at 0.3 mL/min. After the period of 3 loop volumes, gradually increase the flow rate to 1 mL/min. Continue at 1 mL/min until the end of the run and collect 2mL fractions based on the purity of the protein loaded.

    Note: From the SEC run, MIF and D-DT should be eluted as monodispersed peaks. Protein elution may be affected by the buffer composition, loading volume, nature of protein (wild-type or mutant) and column quality/packing (home-packed vs factory made). Therefore, the elution points of MIF and D-DT may vary.

  • f.
    Analyze the purity of the protein fractions by an SDS-PAGE gel.
  • g.
    Concentrate the pure MIF and D-DT sample with a 15 mL Amicon concentrator (10 kDa MW cutoff) at 2100 × g until reaching volumes of 0.5–1 mL.
  • h.
    Check the sample’s concentration using the bicinchoninic acid (BCA) assay.
  • i.
    Make ∼50 μL aliquots in 1.5mL freezer tubes and flash freeze using liquid nitrogen.
  • j.
    Store in a −80°C freezer.

    Pause point: The proteins can be kept at −80°C until ready to be used for the keto-enol tautomerase assay. Aliquots should be thawed on ice and used only once.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains
BL21 (DE3) E. coli/pET-11b with human WT MIF	This study	N/A
BL21 (DE3) E. coli/pET-22b with human WT D-DT	This study	N/A
Chemicals, peptides, and recombinant proteins
4-Hydroxyphenylpyruvate (4-HPP)	TCI	Cat# H0294
Agar	VWR	Cat# J637
Ammonium acetate	VWR	Cat# BDH9204
Ampicillin sodium salt	VWR	Cat# 97061-442
Boric Acid	VWR	Cat# BDH9222
Tris.HCl	Bioland Scientific	Cat# CT02
IPTG	Bioland Scientific	Cat# CI01
LB	VWR	Cat# 97064-114
NaOH	RPI	Cat# S24000
NaCl	N/A	N/A
WT D-DT	This study	N/A
WT MIF	This study	N/A
Glycerol	RPI	Cat# G220254.0
BSA standards	Thermo Fisher	Cat# 23208
Critical commercial assays
Pierce BCA Protein Assay Kit	Thermo Fisher	Cat# 23225
Recombinant DNA
pET-11b - WT MIF	This study	N/A
pET-22b - WT D-DT	This study	N/A
Experimental models: Cell lines
BL21(DE3) competent cells	Agilent Technologies	Cat# 200131
Software and algorithms
GraphPad Prism 9 (used for Figure 2)	GraphPad	https://www.graphpad.com/scientific-software/prism/
BioRender (used for graphical abstract and Figure 1) Note: these figures were created with BioRender.com under the paid subscription.	BioRender	https://www.biorender.com/
Other
0.22 μm PES syringe filter	Bioland Scientific	Cat# SFPE3322S1
0.45 μM PVDF filter	Millipore	Cat# HVLP04700
Microplate reader	Tecan	Model# Infinite M-Plex
Aluminum foil	N/A	N/A
Amicon Concentrator (10 kDa cutoff)	Millipore	Cat# UFC901024
Centrifugation bottles	Thermo Scientific	Cat# 3141-0500
1.5 mL microcentrifugation tubes	Bioland Scientific	Cat# TUBE01-02C
15 mL conical tubes	Bioland Scientific	Cat# TUBE0150-01G
50 mL centrifuge tube	Corning	Cat# 352070
10 μL pipette tips	Bioland Scientific	Cat# TIPS10-11
200 μL pipette tips	Bioland Scientific	Cat# TIPS200-11
1000 μL pipette tips	Bioland Scientific	Cat# TIPS1000-01WC
96-well microplate	Greiner	Cat# 655101
SDS-PAGE Gel	Bioland Scientific	Cat# QP3510
S75 16/600 SEC column	Cytiva	Cat# 28989333
HiTrap Q HP Column	Cytiva	Cat# 17115301
Q-Sepharose XL beads	Cytiva	Cat# 17507201

Materials and equipment

• •MIF Lysis buffer:

Reagent	Final concentration	Amount
Tris.HCl	20 mM	3.15 g
NaCl	20 mM	1.17 g
NaOH (8–10M)	N/A	∼ 0.75–1.25 mL
ddH2O	N/A	∼ 999 mL
Total	20mM Tris.HCl, 20mM NaCl, pH 7.40)	1 L

Note: Filter with a 0.45 μM PVDF filter. Can be stored at 22°C for a week.

• •D-DT lysis buffer:

Reagent	Final concentration	Amount
Tris.HCl	20 mM	3.15 g
NaCl	20 mM	1.17 g
NaOH (8–10M)	N/A	∼ 2–3 mL
ddH2O	N/A	∼ 998 mL
Total	20 mM Tris.HCl, 20 mM NaCl, pH 8.50)	1 L

Note: Filter with a 0.45 μM PVDF filter. Can be stored at 22°C for a week.

• •D-DT elution buffer:

Reagent	Final concentration	Amount
Tris.HCl	20 mM	3.15 g
NaCl	20 mM	58.44 g
NaOH (8–10M)	N/A	∼ 2–3 mL
ddH2O	N/A	∼ 990 mL
Total	20 mM Tris.HCl, 1 M NaCl, pH 8.50)	1 L

Note: Filter with a 0.45 μM PVDF filter. Can be stored at 22°C for a week.

• •Borate solution: add 61.83 g of boric acid to 900 mL of ddH₂O and adjust the pH to 6.2 by adding NaOH and then add ddH₂O up to 1 L.

Can be stored at 22°C for several months.

• •4-HPP buffer: Dissolve 38.54 g of ammonium acetate in 800 mL of ddH₂O. Adjust the pH to 6.20 with acetic acid and fill the solution up to 1 L with ddH₂O.

Can be stored at 22°C for several months.

• •4-HPP solution: Dissolve 27 mg of 4-Hydroxyphenylpyruvate in 5 mL of substrate buffer in a 15 mL conical tube (This will yield a 30 mM final concentration). Wrap the tube well in foil and incubate while rocking for ∼16 h.

Note: The substrate should be >95% purity and the solution should always be made fresh the night before the experiment. Low substrate quality may affect assay results and reproducibility (for more information, see problem 4 in the troubleshooting section).

Cannot be stored.

Step-by-step method details

Prepare the assay plate

Timing: 1 h

In this step, the protein is thawed, and the reaction plate is set up.

Note: Where possible, use a multichannel pipette to increase reproducibility.

• 1.
  Prepare the protein.

  • a.
    Thaw the protein on ice (During this time, step 2 can be performed).
  • b.
    Dilute D-DT to 3.75 μM and MIF to 0.75 μM with their respective lysis buffers.
• 2.
  Prepare the substrate.

  • a.
    Make dilutions of the 4-HPP solution ranging from 0-30mM (This will yield a range 0–2 mM final 4-HPP concentrations).
• 3.
  Prepare the plate (Figure 1).

  • a.
    Pipette 10 μL of the substrate solution into each well (Figure 1 - step 1).

    Note: The color gradient shown in this figure does not represent the actual color you will observe, and it was only used to indicate 4-HPP dilutions. The solutions may appear pale yellow or colorless depending on the 4-HPP sample (most likely colorless).

  • b.
    Introduce 130 μL of borate solution (Figure 1 - step 2).

    Note: If a modulator is going to be used, reduce the volume of borate solution to 128.5 μL and add 1.5 μL of the modulator, dissolved in 100% dimethyl sulfoxide (DMSO). With this approach the final concentration of DMSO remains constant at 1%. Higher DMSO concentration may affect the catalytic activity of MIF and D-DT.

  • c.
    Mix by pipetting.

Figure 1.

A 96-well microplate assay to measure the keto/enol tautomerization activity of MIF and D-DT

The substrate gradient is prepared for each experimental group, in triplicate, and added at a final concentration range of 0–2mM (step 1). Following, 130 μL of borate solution is added (step 2), and the reaction is initiated with 10μL of enzyme (step 3). The total reaction volume is 150μL and the formation of borate-enol HPP is monitored at 306 nm (step 4) over the period of 180s and 300s for MIF and D-DT, respectively.

Perform the assay

Timing: 3–5 min per reaction

Here, the protein is added to the prepared reaction plate and the keto-enol tautomerase activity is monitored.

Note:Methods video S1a and S1b provide an example of how to perform the following steps accurately and reproducibly.

• 4.
  Initiate the reaction.

  • a.
    Pipette 10 μL of the desired protein solution (MIF or D-DT) into a column of the prepared reaction plate (Figure 1 - step 3).
  • b.
    Mix well by pipetting.

    CRITICAL: The mixing needs to be quick, but thorough. The number of times each well is mixed should remain consistent between runs or else the data may not be very consistent. Use low retention tips.

  • c.
    Start readings at 306 nm (Figure 1 - step 4), take readings every 10 s for a total of 180 s (MIF - see Table 1) or 300 s (D-DT- see Table 2).
  • d.
    Repeat steps a-c for all desired replicates/experimental groups. Each experimental group should be tested in triplicate (Figure 1 - see microplate).

    CRITICAL: It is important to only test one column at a time as reading should be taken consistently every 10 seconds. Doing experiments on more than one column may jeopardize the timeliness and consistency of these readings.

Table 1.

Representative experimental absorbances of a MIF kinetic assay in the absence of an inhibitor

4-HPP (mM)	Time (s)
	0	10	20		160	170	180
0	0.1996	0.1999	0.2001	…	0.2011	0.2007	0.2013
0.1	0.2463	0.2558	0.265	…	0.3757	0.3817	0.3887
0.2	0.3099	0.3276	0.3445	…	0.5504	0.5601	0.5729
0.4	0.411	0.4422	0.4722	…	0.8344	0.8536	0.8748
0.8	0.6046	0.6526	0.6995	…	1.269	1.3031	1.3427
1.6	0.9917	1.0622	1.1286	…	1.9134	1.9603	2.0088
2.0	1.1601	1.2356	1.3093	…	2.1211	2.1866	2.2289

Table 2.

Representative experimental absorbances of a D-DT kinetic assay in the absence of an inhibitor

4-HPP (mM)	Time (s)
	0	10	20		280	290	300
0	0.207	0.2071	0.2073	…	0.2096	0.2102	0.2101
0.1	0.2455	0.2477	0.2501	…	0.2958	0.2974	0.2979
0.2	0.2937	0.2965	0.3005	…	0.3816	0.3846	0.3874
0.4	0.3751	0.3821	0.3896	…	0.5382	0.5437	0.5492
0.8	0.5516	0.5646	0.5776	…	0.8365	0.8429	0.8524
1.6	0.9211	0.9385	0.9616	…	1.3869	1.4	1.4152
2.0	1.1262	1.148	1.1714	…	1.6574	1.6725	1.6917

Expected outcomes

Plotting absorbances at 306 nm over time (Tables 1 and 2) will result to a series of straight lines, the slopes of which represent Abs/s (Figure 2A for MIF and Figure 2B for D-DT). To obtain velocities (M/s), divide Abs/s by the molar extinction coefficient of borate-enol HPP (ε₃₀₆ = 11,400 M^-1 cm^-1).⁵ Following, multiply these values by 1 million to get velocities expressed in μM/s. Using GraphPad Prism, fit the data with the Michaelis-Menten nonlinear regression (Figure 2C for MIF and Figure 2D for D-DT). From the Michaelis-Menten curve, one can derive K_M and v_max, and subsequently k_cat. If a modulator is used, the Michaelis-Menten plots can be utilized to extract more information such as the type of inhibition and inhibition constant (K_i). Published examples can be found here ² as well as here.³

Figure 2.

MIF and D-DT kinetic analysis

(A and B) The experimental absorbances shown in Tables 1 (MIF) and Table 2 will be fitted in the linear regression mode of GraphPad Prism to produce a series of straight lines with different slopes (Abs/s).

(C and D) The Michaelis-Menten plots are obtained by converting Abs/s to velocities as described in the protocol (see expected outcomes section).

Quantification and statistical analysis

The tables below provide representative examples of values that should be obtained during a typical MIF or D-DT experiment. Analysis in GraphPad Prism will provide the Michaelis-Menten parameters (Figure 2).

Note: Abs values may be affected by the presence of an inhibitor and/or 4-HPP origin.³

Note: Reported abs values may be affected by the presence of an inhibitor and/or 4-HPP origin.³

Limitations

This protocol requires timely and accurate pipetting (see Methods video S1a and S1b). Utilization of a multichannel pipette is necessary for kinetic and inhibitory experiments as the wells containing the different substrate concentrations need to be initiated and read the same way.

Troubleshooting

Problem 1

Formation of oligomeric MIF/D-DT species during purification.

Potential solution

Formation of MIF/D-DT oligomeric species is usually associated with two factors: i) cleanness of Q-Sepharose column and ii) long retention of the protein in the column. To clean the column, perform multiple rounds of water/high salt (1M NaCl)/water runs, until no contamination comes off the column. Regarding the second factor, fast elution is recommended.

Problem 2

Low protein purity after the Q-Sepharose step. This is applicable to MIF.

Potential solution

Use a longer column or multiple Q-Sepharose columns connected in series.

Problem 3

Poor separation of peaks during SEC.

Potential solution

Poor peak resolution during SEC purification can usually be solved by reducing the flow rate and/or using a higher salt buffer, if necessary.

Problem 4

The rate at which the enol-borate complex is formed is inconsistent or data between trials are not reproducible.

Potential solution

Inconsistent and poor-quality data can often be attributed to stale reagents, poor mixing, or slow pipetting. If 4-HPP powder is left out at 22°C for too long, its stability may decrease. Poor mixing upon reaction initiation is one of the most common mistakes.

Problem 5

Unusually high or low abs readings for individual wells during the assay.

Potential solution

Unusual abs readings at any substrate concentration can be a result of poor mixing, the formation of bubbles or the combination of these two. Mixing with a multichannel pipette should be quick, but thorough. However, using the whole solution volume (150 μL) to mix, will most likely create bubbles. In order to avoid this, it is recommended to use just over half of the solution volume while the pipette must be completely submerged in the well solution.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Georgios Pantouris, gpantouris@pacific.edu.

Materials availability

The plasmid generated in this study is available upon request.

Data and code availability

This protocol includes a representative dataset generated and analyzed during this study.