Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This method can help answer key questions in the drug screening field, such elucidating the effect of small-molecule inhibitors on very low concentrations for protein-protein interactions. The main advantage of this technique is that it is calibration-free, it can be implemented in any confocal microscope, and utilizes very small amounts of labeled proteins. The implications of this technique extend towards development of cancer drugs, small-molecule inhibitors, and various fusion inhibitors because it provides quantitative information on protein-protein interactions.

Though this method can provide insight into protein interactions and aggregations in vitro, it can also be applied to other systems, such as live cell protein dynamics and cell-cell interactions. To begin, transform pLysS cells with a pET-22b vector containing monomerized human FKBP12 and N-terminal His6 and mVenus tags. After the cells have recovered from being incubated and heat-shocked, plate them onto LB agar supplemented with antibiotics.

Transfer the transformed colonies into a 100-milliliter LB starter culture, and grow for 16 to 20 hours at 37 degrees Celsius with shaking. Following incubation, dilute the dense starter culture one to 100 in LB medium in two 500-milliliter batches. Then, grow for two to three hours to an optical density at 600 nanometers of 0.6 to 0.8.

After cooling the cultures on ice, induce protein production with 250-micromolar IPTG. Grow the cultures for 16 to 20 hours at 21 degrees Celsius and 200 rpm. Then, harvest cells by centrifugation at 2, 000 g for 20 minutes.

Remove the supernatant, and resuspend the pellet in 40 milliliters of IMAC buffer A supplemented with EDTA-free protease inhibitors. Now, sonicate the cells at 500 watts, 20 kilohertz, and 40%amplitude at nine seconds on, 11 seconds off for 15 minutes on ice. Following sonication, harvest the soluble material by centrifugation at 20, 000 g for an hour at four degrees Celsius.

Transfer soluble lysate to a conical flask, and add two milliliters of TALON resin. Allow the suspension to incubate for one hour with 105 rpm rotation. Now, harvest the resin, and wash it with 250 milliliters of IMAC buffer A followed by 500 milliliters of IMAC buffer B.Elute His6-tagged protein using IMAC buffer C.Inject the eluate onto a pre-equilibrated size-exclusion column, and collect the FKBP12 peak, which elutes at approximately 87.7 milliliters.

Assess the purity of the protein via SDS-PAGE, and pool and concentrate as required. To set up the multiwell plate array, first prepare a solution of 100-nanomolar purified FKBP12 in the same buffer used for size-exclusion chromatography. Sonicate and centrifuge with a quick spin of 13, 000 rpm to prevent the formation of aggregates.

Now, pipette 100 to 200 microliters of the diluted protein into an eight-well observation chamber with a glass bottom. Add the BB dimerizer to final concentrations of 10, 20, 40, 80, 100, 150, 300, and 500 nanomolar. As a reference, prepare a solution of 100-nanomolar mVenus alone to evaluate potential aggregation and precipitation effects and to recover a brightness value for the monomer with the same acquisition settings.

Any light scanning microscope confocal system equipped with digital detectors or well-characterized analog detectors and capable of keeping a constant dwell time for every pixel acquired can be used. Select the collar correction water immersion objective designed for fluorescence correlation spectroscopy. Now, add a drop of water to the collar correction water immersion objective.

Mount the eight-well observation chamber into the stage. Set the excitation beam path by turning on the 514-nanometer laser and setting it at 20 to 100 nanowatts power at the exit of the objective. Turn on one HyD detector.

Detectors capable of photon-counting are preferable. Select the emission window from 520 to 560 nanometers. For the acquisition mode, use 16 by 16 pixels.

Set the pixel dwell time such that the frame time is longer than the protein diffusion and the pixel dwell time is much shorter. This corresponded to setting the dwell time to approximately 13 microseconds for the system used in this demonstration. Set the pinhole at one Airy unit for the corresponding emission of approximately 545 nanometers.

Select the xy time acquisition mode, and select the number of frames to be acquired per acquisition and well. Now, set the pixel size at approximately 120 nanometers. If the system is equipped with high-throughput mode, introduce the coordinates of each well and the number of acquisitions per well to automate the process.

If the system is equipped with a perfusion system, load the BB solution and program the perfusion to start right after the 5, 000th frame to evaluate the kinetics of dimerization while acquiring 10, 000 images. Select the correct well, and focus on the solution. Then, start the acquisition, and save the resulting stack of images in TIFF format.

Sequences of 20, 000 images of purified FKBP12 mVenus in 100-nanometer solution were acquired as specified in the protocol section. The intensity of the first frame is shown together with the mean intensity profile of the detrended image. The brightness without and with smooth filtering is also shown.

After 10, 000 frames were acquired, the homodimerizer drug BB was added to the solution while acquiring. Each consecutive series of 5, 000 frames was analyzed. The mean brightness five minutes after BB addition was 1.010, which is a twofold increase, indicating an FKBP12 dimer.

The kinetics of the process shows a delay between BB addition and full FKBP12 dimerization of approximately two minutes. A key aspect to evaluate protein-protein interactions in vitro is the production and purification of your labeled protein. Be sure you have small amounts of the labeled protein of interest and that your protein is not aggregating per your analysis.

Following this procedure, other methods like surface plasmon resonance or fluorescence correlation spectroscopy can be performed in order to answer additional questions, like dissociation constants or diffusion coefficients. After its development, this technique paved the way for researchers in the field of biophysics to explore in detail protein-protein interactions in live cells. Don’t forget that working with reagents for protein purification can be extremely hazardous, and precautions, such as safety glasses, gloves, should always be taken while performing this procedure.