Experiments

In this section, we describe the experiments that we have performed and those planned to test the protein inhibitors designed through protein modeling. To learn more about how the minibinders were designed, please visit our Engineering Success page. We conducted protein inhibitor purification, mutated and recombinant Exotoxin A purification, LRP1 immunofluorescence, and western blot experiments. We also plan to perform future experiments, including SPR, flow cytometry, LDH and MTT cytotoxicity assays, SUnSET, and fixed-cell fluorescence assays. Here, we use the terms protein inhibitor, miniprotein, and minibinder interchangeably. Use the left sidebar to jump to specific sections for easy reference.

We purified the minibinders, proteins we designed to inhibit Exotoxin A, in preparation for future experiments to test their binding to Exotoxin A.

Golden Gate Assembly and Transformation

Gene fragments encoding the miniproteins were bought from Twist Bioscience and IDT. Each gene fragment contained a BsaI site, which allowed for insertion into the plasmid LM627 using Golden Gate Assembly. In this method, the BsaI restriction enzyme cuts outside of its recognition sequence, generating overhangs. The overhangs were designed so that the gene fragment and the plasmid can fit together. T4 DNA Ligase puts the plasmid and the gene fragment together.

The assembled plasmids were then transformed into BL21(DE3) E. coli by heat shock. After the transformation, the E. coli were grown overnight and then induced with ZYM-5052 auto-induction medium. Because the proteins are under a T7 promoter, the miniproteins were expressed.

Chemical Lysis

Afterwards, the cells were lysed using Bug Buster and lysozyme. They were centrifuged so that the cell debris settles at the bottom and the supernatant with the soluble proteins is at the top. The supernatant was then extracted and loaded into the protein purification column.

His Tag Protein Purification

The proteins were purified with Nickel affinity chromatography with a His Tag. To do this, the His Tag column was prepared. Ni-NTA resin is added, washed with MilliQ water, and equilibrated with NiSO₄. To allow buffers and samples to be pulled through a centrifuge or vacuum manifold was required. The lysate was then poured into the columns and sat for 10 minutes. It was then pulled through, which removed the unbound proteins, DNA, and RNA. The column was washed with His Wash Buffer, which contains low concentrations of imidazole. The flow-through contains weakly interacting proteins with the Nickel resin. His Tag proteins were eluted with Elution Buffer, which had high concentrations of imidazole.

High Performance Liquid Chromatography (HPLC)

Finally, the proteins were purified with size exclusion chromatography (SEC) using HPLC to remove aggregated proteins. Samples from the 96-well plate were loaded into the HPLC system. The pump pushed the solvent or the mobile phase through the HPLC column. The purified protein from the His-Tag Purification was pushed through the column at the same time. The column is packed with porous beads, which allows the separation to take place. Large molecules move around the beads eluting first, while small molecules move through the small holes in the beads, bumping around and eluting last.

These samples were separated and detected with a UV absorbance detector. The detector sends this information to the computer and generates a plot of intensity over time. Each peak represents a separate component in the sample, and the peak area corresponds to the concentration of the component. Aromatic acids (tryptophan, tyrosine, phenylalanine) absorb strongly at 280 nm, and an aromatic amino acid was placed on the protein via the plasmid we chose, LM627. After detection, the mobile phase and the analytes are collected as waste. Samples with the protein were normalized to the same final concentration.

Objective: To detect and quantify ExoA proteins in HEK293T cells by separating them by size and probing with antibodies.

HEK293T Transfection

HEK293T cells are a type of human kidney cell that is commonly used in labs due to their durable nature and their ease in taking up plasmids and expressing the desired proteins. We expressed LRP1 in HEK293T cells, so that we could have high expression of LRP1 in cells. We want to use these cells to test the minibinders and see if they block Exotoxin A from binding to LRP1.

HEK293T cells were plated evenly in a plate in complete media. For a 6 cm plate, 1.4 million cells were used, and for a 10 cm plate, 4 million cells were used. After 18–24 hours, the cells reattach to the plate and recover from the plating process. Cells were then transfected with plasmid constructs using polyethylenimine (PEI) as the transfection reagent. PEI helps DNA enter by binding to DNA and then being taken up by the cell. A DNA:PEI ratio of 1:3 was maintained, with 1–6 µg of each construct added to 500 µL of Opti-MEM per plate. Cells were then incubated at 37 °C with 5 % CO₂ to allow protein expression.

After 48 hours of expression, the cells were lysed with lysis buffer and placed in a shaker at 4 °C to fully lyse the cells. The samples were then centrifuged in a refrigerated centrifuge to separate cell debris from the soluble proteins and to preserve protein and enzyme stability at low temperatures. We moved the lysate to a separate tube and measured the protein concentration using a bicinchoninic acid (BCA) assay. The BCA assay is a colorimetric method that utilizes a chemical reaction between peptide bonds and copper ions in a basic environment. This reaction turns the sample purple and strongly absorbs light at 562 nm. A standard curve was generated using six protein standards (0, 2, 4, 8, and 16 µg/mL), and the absorbance of the unknown samples was measured to determine their protein concentrations.

The protein samples were then mixed with LDS and DTT and lysis buffer to keep the protein stable. LDS denatures proteins, and DTT breaks bonds. This makes the proteins stable and able to be stored for a long time in −20 °C. Protein samples are then heated at 37 °C for an hour to complete protein denaturation.

Proteins are separated based on molecular weight using a 10 % Nu-PAGE precast gel. Smaller proteins move faster through the gel while larger proteins move more slowly.

After electrophoresis, the proteins were transferred from the gel to a membrane. They were transferred for 3 hours at 4 °C at 300 mA. The membrane paper serves as a place for antibody detection of proteins. It was blocked in 5 % milk prepared in TBST (1×TBS + 0.1 % Tween). Blocking prevents non-specific binding of antibodies and allows for less background noise. Milk occupies non-specific sites, while Tween, a detergent, prevents non-specific binding.

It was then incubated overnight with primary antibodies, and then for about an hour with secondary antibodies with a fluorescent tag. They were then imaged to see if there were proteins in the expected spot (~660 kDa) on the western blot.

We used fluorescently labeled secondary antibodies to detect expression of LRP1 protein in our human embryonic kidney cells (HEK293T).

In order to validate the expression of LRP1 in our human embryonic kidney cells (HEK293T), we run an immunofluorescence assay (or immunocytochemistry) protocol. Preparing this protocol begins by plating and culturing HEK293T cells to a healthy confluence, typically on a glass coverslip. We then remove them from their sterile environment and fix them with paraformaldehyde. These fixed cells are stored at 4° C.

After preparation, we run the immunofluorescence assay by permeabilizing the membranes of fixed cells with mild detergent, then applying the two antibodies in a series. First, the primary antibody is applied to the permeabilized cells targeting the LRP1 protein, and left to incubate. After incubation, the primary antibody is washed away with PBS or PBST and a secondary antibody is applied. The secondary antibody is conjugated with a fluorophore, and it binds to the primary antibody. After the final washing, which removes any unbound secondary antibody, the coverslips are ready to be loaded onto the slides for imaging.

The combined treatment of primary and secondary antibodies allows for the labeling of specific proteins of interest. In our case, we use it to confirm the presence of membrane protein LRP1 after its transfection to validate our large protein transfection protocol.

We purified both the mutated, nontoxic ExoA and the recombinant, nontoxic ExoA. To see the gene fragments we used, please visit our Parts Collection page. The mutated ExoA will be used to test our minibinders using SPR, while the recombinant ExoA will be used to test the minibinders through flow cytometry or fixed-cell fluorescence assays.

We bought gene fragments from GenScript encoding Exotoxin A: Domain 1a, a truncated form of Domain 1a, full-length Domain 1a, full-length Exotoxin A with mutations, and Domain 1 and 2 fused with two fluorophores. We assembled these fragments using Golden Gate Assembly. We did this by mixing the vector, the gene fragment, T4 DNA ligase, the BsaI enzyme, T4 DNA ligase buffer, and nuclease-free water in a reaction. This ligated the gene fragment into the plasmid LM1425. The gene fragments were specially designed to have overhangs that match those of LM1425, so when BsaI cut the fragments, they aligned perfectly with T4 ligase.

After the Golden Gate Assembly, the plasmids were transformed into E. coli. The cells were first incubated, then heat-shocked for 30 seconds and placed back on ice. After that, SOC media was added to help the cells recover and incorporate the plasmid properly. They were then grown in SOC media for about an hour. Following recovery, the cells were plated onto kanamycin-containing plates and streaked to obtain colonies. The plates were incubated overnight at 37°C.

Once colonies grew, we performed PCR for sequencing using forward and reverse primers associated with plasmid, LM1425. The PCR products were sent for sequencing, and we received the results the next day. Looking at the sequences, we confirmed that the sequences were correct.

After sequence verification, we saved glycerol stocks for long-term storage in the freezer. At this point, we did not continue with further experiments due to time constraints before the wiki freeze. The next steps would involve growing the cells overnight in a 4mL tube with TB and Kanamycin. They would then be transferred to autoclaved 50mL flasks with TB II and autoinduction media and grown overnight.

After expression, cells would be centrifuged for 10–12 minutes and the supernatant removed. The cell pellet could be frozen at −20°C overnight or resuspended in lysis buffer. Because Exotoxin A forms inclusion bodies, it would need to be denatured. This is done by preparing the lysis, elution, and wash buffers with 6 M guanidinium chloride and 1 mM TCEP. Guanidinium chloride denatures proteins, and TCEP reduces disulfide bonds. Cells would then be lysed with a sonicator, and the lysate centrifuged. The pellet can be retained until SDS-PAGE is performed to confirm protein expression. All centrifugation should be done at refrigerated temperatures.

His-tag purification columns would be equilibrated, the lysate applied, incubated for 10 minutes, and washed. Proteins would then be eluted with an elution buffer containing a high imidazole concentration. Because the proteins are denatured and purified with His Tag purification, they need to be dialyzed to gradually refold the proteins. They would then be purified through SEC-HPLC.

Planning for the Future

Below are experiments currently under consideration and planned for the future. These include SPR, Flow Cytometry, LDH, MTT, SUnSET, and Endocytosis Timecourse assays. These do not have notebook entries as they have not been done.

Surface Plasmon Resonance

Surface Plasmon Resonance is a biochemical technique that measures the binding affinity between two proteins. It detects changes in reflective light at the surface of a sensor chip, which occur when molecules bind to proteins immobilized on the chip.

We wanted to use SPR in two ways:

1. Testing ExoA folding (including fluorophore fusions):
To purify mutated ExoA, we have to express, denature, purify, and then refold the proteins. Considering that this is a difficult process, we needed to check if it worked using SPR. We would use SPR by adding an AviTag to ExoA and immobilizing it on the sensor chip. Then we could pass over an antibody that binds ExoA, or LRP1, which ExoA is known to bind based on previous studies.

To express ExoA, we used a plasmid that already contains an AviTag, so we only needed to biotinylate the purified ExoA before using it for SPR. This setup would allow us to measure the binding affinity. If ExoA binds to LRP1 or the antibody, it would indicate that it is folded properly. If it is misfolded, there would be little or no binding. This gives a direct, functional readout of correct folding.

2. Testing minibinder interactions:
SPR can also be used to test whether our minibinders bind to ExoA. After confirming that ExoA is properly purified and folded, we could use SPR to test minibinder binding. This same approach could also be used with ExoA, with ExoA being biotinylated and immobilized on the sensor chip, and the minibinders passed over it.

General SPR workflow

The first step in SPR is to prepare different dilutions of the minibinder or antibodies to measure binding affinity. If lower concentrations show weaker binding while higher concentrations show stronger binding, it indicates proper binding behavior. The next steps involve preparing the biotin capture reagent and the regeneration reagent, which is used between runs. Then, the SPR buffer and sensor chip are changed and correctly placed. The sample is loaded into the plate, and the run is started. Afterwards, the screening data can be analyzed to evaluate binding.

During the run, the biotinylated ligand is pushed over the sensor chip surface. When it flows past the chip, the biotin binds to streptavidin. The excess ligand is washed away and it leaves a uniform layer of the exotoxin A on the chip. After that the the mini binders or the antibodies are injected and flow over the sensorship. If binding occurs, more mass is on the surface of the chip, and as a result, the light that passes over the sensor chip changes its reflectiveness. A regeneration buffer removes the minibinders and without stripping off the biotinylated ligand as a result it's the chip is restored to its original baseline. The cycle and testing of minibinders against the chip can be repeated.

During the experiment, the instrument records a sensorgram, which is a graph that shows how binding occurs over time, as you can see in the image below. The x-axis represents time, and the y-axis represents response units. When the minibinder or antibody binds to ExoA, the mass on the chip increases, causing the response units to rise. This change is detected by how much the reflected light shifts. When the minibinder or antibody is first flowed over the chip, the curve begins to rise as binding occurs. This is the association phase. Once the sample flow stops and only the buffer is passed over the chip, the bound molecules begin to detach from ExoA, leading to a gradual decrease in the response signal. This downward part of the curve is called the dissociation phase. The slope of the curve provides information about how quickly the molecules bind and unbind. This can then be used to calculate their binding kinetics and affinity.

This is very valuable because it allows mobilization at a specific site on the exotoxin A. It also enables accurate measurement of kinetics and affinity for protein-protein interactions.

We quantified extracellular protein uptake and lysosomal degradation using ExoA fusion constructs, flow cytometry, and immunoblotting.

To test and quantify the effectiveness of our inhibitors, we designed a double protein fusion of Exotoxin A - mCherry and sfGFP. After ExoA binds to LRP1 and is subsequently endocytosed, sfGFP rapidly degrades in the lysosome while mCherry remains stable in a lower pH. Thus, the uptake can be tracked by the portion of cells with loss of green fluorescence against a stable red signal.

Recombinant double fusion proteins are created by concatenating DNA sequences for Exotoxin A domain 1 (the LRP1 binding domain) with mCherry and sfGFP, with flexible linkers in between. These fluorescent proteins are produced according to the same protocol for ExoA purification.

With these proteins in hand, we apply them in a 24-hour time period to LRP1-expressing cells, these cells can be either our HEP-G2 LRP1-positive controls, our transduced HEK293T LRP1 model cell lines, or our stem cell-derived cardiac fibroblast model. During this incubation period, separate portions (wells) of the cells can be tested with our protein minibinders meant to prevent ExoA endocytosis.

After the incubation period, flow cytometry is used to rapidly measure fluorescence in thousands of cells from each group. Gating for the fluorescence readout is done as described above, with cells that display red but not green fluorescence understood to have internalized the exotoxin. The quality of minibinders is then assessed by their ability to reduce the portion of cells with exotoxin A uptake.

As a validation step for the internalization assay, we perform immunoblot analysis on a lysis of the cell cultures. After washing cells multiple times with PBS to remove extracellular ExoA fusion protein, we harvest and lyse the cells for their protein. We run this protein on a western blot assay using an anti-ExoA antibody to confirm the successful uptake of ExoA into the cells.

Protocol adapted from a clusterin internalization protocol:
A. Tomihari, M. Chiba, A. Matsuura, and E. Itakura, “Protocol for quantification of the lysosomal degradation of extracellular proteins into mammalian cells,” STAR Protocols, vol. 2, no. 4, p. 100927, Nov. 2021, doi: 10.1016/j.xpro.2021.100927. [Online]. Available: https://star-protocols.cell.com/protocols/1197#step-by-step-method-details

LDH and MTT Cytotoxicity Assay

Lactate dehydrogenase (LDH) is a cytosolic enzyme present in many different types of cells. When the plasma membrane is damaged, LDH is released into the cell culture media. The released LDH can be quantified by a coupled enzymatic reaction. First, LDH catalyzes the conversion of lactate to pyruvate via the reduction of NAD⁺ to NADH. Second, diaphorase uses NADH to reduce a tetrazolium salt (INT) to a red formazan product. Therefore, the level of formazan formation is directly proportional to the amount of released LDH in the medium.

In our system, HepG2 cells endogenously express LRP1, the receptor that mediates uptake of Pseudomonas aeruginosa Exotoxin A (ExoA). ExoA inhibits protein synthesis by ADP-ribosylating EF-2, which leads to loss of viability and eventual membrane damage.

First, we establish a sub-lethal ExoA dose in HepG2 cells. We plate cells and run an ExoA-only titration in parallel with LDH and MTT assays across a log-spaced dose range and one or more time points. We select a working dose where MTT viability is reduced but not collapsed and LDH is elevated above baseline but not near the lysis control. This gives dynamic range to detect protection by binders.

We then use LDH release to read out membrane integrity after ExoA exposure and to test whether our designed mini-protein binders block toxin entry or action. Percent cytotoxicity is calculated by normalizing the sample LDH to the spontaneous and maximum controls. Binder efficacy is reported as percent protection relative to the ExoA-only condition and by shifts in ExoA EC₅₀ in the presence of binder.

As with LDH, we begin by determining a sub-lethal ExoA dose in HepG2 using an ExoA-only titration read by both MTT and LDH. We pick a dose and time point where MTT shows a clear drop from baseline but retains headroom for rescue, and LDH has not plateaued.

The MTT assay measures metabolic activity as a proxy for viable cell number. Viable cells reduce the yellow tetrazolium MTT to insoluble purple formazan crystals that are solubilized and read at ~570 nm. For ExoA studies, MTT detects early loss of metabolic activity due to translation arrest before extensive membrane rupture. We run matched conditions to LDH: untreated, vehicle, ExoA only at the selected sub-lethal dose, binder only, and binder+ExoA dose matrices. Increased MTT signal in binder+ExoA wells relative to ExoA only indicates protection. Reporting includes percent viability normalized to untreated controls and estimates of binder potency across ExoA doses. Using MTT together with LDH lets us distinguish early metabolic decline from later membrane damage and strengthens conclusions about mini-binder rescue.

We first determine a sub-lethal ExoA dose in HepG2 cells by running ExoA-only titrations read out with LDH and MTT, then select a dose and time point that reduces viability without collapsing the signals. At that working dose, we quantify global translation with SUnSET, a non-radioactive puromycin-based assay. Puromycin is incorporated into nascent polypeptides by the ribosome, and anti-puromycin antibodies detect these tagged chains by immunoblot, immunofluorescence, or flow cytometry. Signal intensity tracks ongoing protein synthesis without radioactive labeling.

Because ExoA ADP-ribosylates eEF2 and shuts down elongation, ExoA decreases puromycin incorporation. Protection by mini-binders is indicated by restoration of the puromycin signal toward untreated levels. Include untreated, vehicle, ExoA-only at the chosen sub-lethal dose, binder-only, and binder+ExoA conditions across binder:toxin ratios. Cycloheximide can serve as a translation-block positive control to confirm dynamic range and antibody performance. Report percent translation relative to untreated cells and shifts in the ExoA inhibition curve in the presence of the binder.

Reference method: SUnSET was introduced as a simple alternative to radioactive metabolic labeling and has since been applied widely across cell types and tissues.

In this assay, we plot a time course of increased fluorescence in our LRP1-expressing cells due to the uptake of fluorescent Exotoxin A. In order to run the assay, we first run a titration by plotting a time course of fluorescence at various concentrations of our ExoA fluorescent fusion protein. By examining the results of the titration, we'll select a concentration and time parameters that produce a smooth, steady increase in fluorescence to run the assay with. We'll plan to run the assay at a single concentration with several timepoints for readout.

With the parameters set for the assay, we can continue to the trials with our minibinders. We'll run a negative control time course with only Exotoxin A, and a positive control course with the known antibody inhibitor. We'll run various of our candidate minibinders in courses alongside these, and look for successful inhibition of fluorescence compared to the negative control as well as to the known inhibitor positive control.

Notable about this experiment is that due to the high number of cells and wells needed for this experiment ([# of minibinders + 2 controls] * # of timepoints = number of wells), this assay will have a lower throughput than our other eukaryotic cell assays, such as Cytotoxicity or the Flow Cytometry assays. Due to this constraint, it should be run after the higher throughput assays to further examine a smaller selection of more promising minibinders.

Citations

All images not cited are made with Biorender.

1. S. Aryal, “HPLC: Principle, Parts, Types, Uses, Diagram,” Microbe Notes, May 24, 2024. [Online]. Available: https://microbenotes.com/high-performance-liquid-chromatography-hplc/
2. A. Tomihari, M. Chiba, A. Matsuura, and E. Itakura, “Protocol for quantification of the lysosomal degradation of extracellular proteins in mammalian cells,” STAR Protocols, vol. 2, no. 4, p. 100927, Nov. 2021, doi: 10.1016/j.xpro.2021.100927. [Online]. Available: https://star-protocols.cell.com/protocols/1197#step-by-step-method-details
3. Cell Biologics, LDH Assay – For Research Use Only. Chicago, IL: Cell Biologics, 2017. [Online]. Available: https://cellbiologics.com/document/1495130108.pdf
4. ResearchTweet, “MTT Assay Protocol for Cell Viability and Proliferation,” ResearchTweet, 2021. [Online]. Available: https://researchtweet.com/mtt-assay-protocol-for-cell-viability/