精製と TRPV1 の分光学的解析のための再構成

Here in the lab, we think that using a spectroscopic approach can help answering key questions in the transient receptor potential ion channel family. Such as determining the dynamic informational changes associated with polymodal activation. The main advantage of this protocol is that allow milligram of pure and functional remaining ion channel for electron paramagnetic resonance and double electron electron resonance epitroscopy.

This method can provide insight into the conformational changes compatible with thermal and light independent gating of 3B1. It can also be applied to other trip channels, like 3B2, for example. Begin this procedure with design mutagenesis primers with online tools.

Prepare the PCR reaction as described in the text protocol and spin the mixture. Place the tubes in the thermocycler and perform PCR mutagenesis using the PCR parameters listed in the text protocol. Then add two microliters of DPN1 enzyme to the reaction and mix.

Incubate the reactions at 37 degrees Celsius for 30 minutes. Next, perform the transformation by adding 75 microliters of thawed E.Coli competent cells and 10 microliters of DPN1 treated PCR mixture to a pre-cooled 14 milliliter sterile tube. After mixing gently, incubate reaction on ice for 30 minutes.

Maintain the tube at 42 degrees Celsius for 45 seconds and then immediately place it on ice for two minutes. Plate the mixture on an LB agar plate containing carbenicillin, and incubate overnight at 37 degrees Celsius. After harvesting single colonies and preparing overnights as described in the text protocol, spin down overnight cultures at 8000 times G for 10 minutes.

Then, extract the DNA following the instructions of commercial available mini preparation kits. To generate the recombinant bacmid, transform 100 microliters of DH10bac E.Coli competent cells with 7.5 nanograms of the recombinant donor vector. Vectors contain the functional minimal cysteine-less rat TRPV1, abbreviated eTRPV1, and or single cysteine mutants.

After seeding and incubating cells as described in the text protocol, select white colonies that are two millimeters in diameter since blue colonies lack the insert of interest. Use a stereo microscope to verify that white colonies do not contain any blue spots. Harvest at least three single colonies and transfer them into a 14 milliliter steril tube containing four milliliters supplemented LB media.

Incubate the cells overnight at 37 degrees Celsius in 250 RPM. Take 1.5 milliliters of the overnight cultures and isolate recombinant bacmid DNA. To generate the recombinant baculovirus, plate one million SF9 cells in two millimeters of insect cell media.

Allow the cells to attach for at least 45 minutes. After washing once with one milliliter of fresh medium, remove the wash medium and add DNA transfection reagent mixture to the cells. Incubate the cells at 27 degrees Celsius for five hours without agitation.

Following incubation, remove the transfection mixture and replace with two milliliters of insect cell media containing 0.5%fetal bovine serum. Then, incubate the cells at 27 degrees Celsius for five days. Transfer the cell culture media into a 15 milliliter centrifuge tube.

Isolate the first passage of viral stock by spinning the tube at 8000 times G for 15 minutes at 4 degrees Celsius. Transfer the supernatant to a clean 15 milliliter centrifuge tube and immediately store it at four degrees Celsius. Of the second generation of viral stock, pipette a 25 milliliter suspension culture of insect cell media at one million SF9 cells per milliliter containing 2%FBS into a 125 milliliter flask.

Add 25 microliters of the P1 virus to the 25 milliliter culture. Then, incubate the culture at 27 degrees Celsius for 72 hours. To harvest the P2 viral stock transfer the culture to a new 50 milliliter tube and centrifuge it at 8000 times G for 15 minutes at four degrees Celsius.

Transfer the supernatant to a new 50 milliliter tube and immediately store it at four degrees Celsius. Add one milliliter of P2 virus stock to the one liter culture and incubate it at 27 degrees Celsius for 72 hours. Harvest the one liter insect cell suspension culture by spinning at 4500 G for 20 minutes at four degrees Celsius.

Weigh the cell pellet. It is expected to be around six to nine grams. Resuspend the cell pellet in 25 milliliters of ice cold buffer A in the presence of protease inhibitors.

Disaggregate cell clumps to obtain a homogenous suspension and rotate at four degrees Celsius for 20 minutes. Break the cells using a manual or a high-pressure homogenizer. After removing the cell debris by centrifugation at 8000 times G for 20 minutes a four degrees Celsius, collect the supernatant and centrifuge again at 100, 000 times G for 30 minutes at four degrees Celsius.

Following centrifugation, discard the supernatant and suspend the membrane pellet in a total volume of 20 milliliters of ice cold buffer B supplemented with protease inhibitors. Aliquot 20 milliliters of membrane suspension to 50 milliliter tubes. To perform the protein purification add three milliliters of DDM per tube.

Rotate the tubes for two hours at four degrees Celsius. After centrifuging the sample at 100, 000 times G for 30 minutes at four degrees Celsius, collect the supernatant and add one milliliter of clean, wet amylose resin. Rotate the mixture for two hours at four degrees Celsius.

Then load the protein amylose mixture in gravity flow chromatography columns. Wash the protein bound amylose resin with 10 times the bed volume of ice cold buffer C, followed by 10 bed volumes of ice cold buffer D.Elute eTRPV1 protein with 0.5 milliliter fractions up to five milliliters of ice cold degassed buffer D supplemented with 20 millimolar maltose. Perform the washes and elution at four degrees Celsius.

Using a 100 kilodalton cutoff centrifugal filter unit, concentrate the eTRPV1 containing fractions to between two and 2.5 milligrams per milliliter for labeling. Next, add a ten-fold molar excess of MTSSL spin label from a 100 millimolar stock solution in DMSO to the concentrated protein. Repeat this twice every 30 minutes.

Keep the reaction in the dark at room temperature for one hour and 30 minutes, followed by overnight incubation at four degrees Celsius. Then, load the spin label to eTRPV1 on a size-exclusion chromatography column equilibrated in buffer E, controlled by a fast protein liquid chromatography system. Collect the fractions containing eTRPV1 tetramer.

Dry 10 milligrams of asolectin using a rotary evaporator under vacuum for one hour at 40 degrees Celsius. To make asolectin liposomes, add one milliliter of buffer F and sonicate the mixture for 15 minutes or until the sample is homogenous. Destabilize the liposomes by adding DDM to a final concentration of two millimolar and incubate for 30 minutes at room temperature.

After adjusting the mixture to the DDM critical mycel concentration, as detailed in the text protocol, double the volume of the protein liposome mixture with buffer F.Remove the detergent by sequentially adding three aliquots of non-polar polystyrene absorbent beads at one hour intervals with gentle agitation at room temperature. Load the mixture onto a column filter to remove the beads and transfer the proteoliposome mixture to an ultra-centrifuge tube. Centrifuge the samples at 100, 000 times G for one hour at four degrees Celsius.

Discard the supernatant and resuspend the pellet in 30 microliters of buffer F, and then perform spectroscopic measurements and analysis. The first checkpoint is the functional analysis of single-cysteine mutants before undertaking expression and purification protocols. Here, hek293 cells expressing wild type TRPV1, eTRPV1, and mutants were analyzed for capsaicin evoked responses using florescence calcium imaging.

The second checkpoint is the biochemical characterization of spin labeled single-cysteine mutants through size-exclusion chromatography before proceeding with reconstitution and spectroscopic analysis. The third checkpoint is the functional characterization of reconstituted spin labeled single-cysteine mutants before undertaking spectroscopic analysis. Current voltage relationships determined by two electrode voltage clamp from Xenopus oocytes micro-injected with proteoliposomes containing spin labeled A702C were challenged with pH5 and blocked by capsazepine.

Finally, it is possible to obtain the mobilities of spin labeled eTRPV1 mutants determined by continuous wave EPR. The first derivative spectra are shown for spin labeled cysteine mutants in DDM solution, and reconstituted in asolectin liposomes. Distances and mobilities of spin labeled eTRPV1 mutants in detergent were also determined by DEER.

DEER echo and distance distribution of spin labeled mutant E651C is shown. After watching this video, you should have a good understanding of how to express, beautify, and reconstitute a mammalian ion channel for spectroscopic analysis. Generally, individuals new to this method will struggle because of the lack of protein function when removing cysteine residues, low protein yield, and protein instability either in purification and after spin labeling, and protein aggregation in detergent or liposomes.

After its development, these protocols will allow obtaining distance and dynamic changes to explore mechanistic models for TRPV1 polymodal gating.