효율적인 Refolding 및 Chemoreceptor Ligand 바인딩 도메인의 정화 방법

The overall goal of this procedure is to refold a chemoreceptor ligand binding domain from inclusion bodies and purify it for use in structural and functional studies. To establish what signals a bacterial chemoreceptor sends and how, one can use the isolated ligand binding domains to screen against small molecule libraries or to determine the structure with and without ligand. Epression of extracellular ligand binding domain in E.coli often results in the deposition in inclusion bodies.

Here, we show how milligrams amount of functional protein can be recovered from the inclusion bodies. To begin, inoculate 150 mL of sterile LB broth containing 50 micrograms per mL of ampicillin with BL21-CodonPlus RIPL cells transformed with the pET151 D-TOPO vector for expression of hexahistidine TIp3-LBD. Incubate the culture at 200 RPM and 37 degrees Celsius overnight.

Prepare six 2L Erlenmeyer flasks containing 800 mL of sterile LB broth and 50 micrograms per mL of ampicillin. Inoculate each flask with 20 mL of the overnight culture. Incubate the flasks at 37 degrees Celsius with continuous shaking at 200 RPM until the OD600 reaches 0.6.

At this point, induce protein expression by adding 1 millimolar IPTG to each flask. Continue incubating the flasks at 37 degrees Celsius in a shaker at 200 RPM for an additional four hours. Harvest the cells by centrifugation at 5000 xg and four degrees Celsius for 15 minutes.

Then, discard the supernatant. Transfer all the cell pellets to a 250 mL beaker and add 100 mL of Buffer A.If frozen, allow the cells to thaw completely and then resuspend the pellet. Keep the sample on ice unless indicated otherwise.

Next, pass the resuspended cells through a high-pressure homogenizer three times to lyse the cells and ensure complete shearing of the genomic DNA. Then, centrifuge the lysate at 10, 000 xg and four degrees Celsius for 15 minutes. Collect a one mL sample of the supernatant and store it at minus 20 degrees Celsius for subsequent SDS-PAGE analysis.

Then, discard the rest of the supernatant and place the pellet on ice. Next, thoroughly resuspend the inclusion bodies pellet in 20 mL of ice cold Buffer B.This facilitates solubilization of the membrane and membrane proteins. Vortex the sample for one to two minutes to aid in resuspension.

After centrifuging the sample, thoroughly resuspend the pellet again in 20 mL of ice cold Buffer B by first vortexting the tube for one to two minutes. Ensure that the pellet is broken into small pieces. Then, pipette the sample up and down to resuspend it.

Centrifuge the sample as before and discard the supernatant. If the supernatant is cloudy or colored, centrifuge the sample again and use additional ice cold Buffer B to resuspend it. Repeat the centrifugation and resuspension until the supernatant is clear and colorless before pelleting again.

Add 20 mL of ice cold Buffer C to the pellet and resuspend it by vortexing the tube for one to two minutes. Then, after spinning the sample, add 25 mL of ice cold denaturing Buffer D.Thoroughly resuspend the inclusion bodies pellet by vortexing the tube for one to two minutes or until the pellet is broken into small pieces. Mix the suspension by axial rotation at 30 RPM and 4 degrees Celsius for 30 to 120 minutes.

Clarify the denatured protein solution by centrifugation at 30, 000 xg and four degrees Celsius for 30 minutes. Then, place the supernatant on ice and discard the pellet. While stirring 250 mL of Buffer E at 500 RPM, add 60 milligrams of denatured protein mix containing hexahistadine TIp3-LBD.

Incubate the refolding mix at four degrees Celsius with continuous stirring at 500 RPM for 24 to 48 hours. The final protein concentration is 0.2 mg per mL. After preparing seven L of pre-cooled Buffer A and dialysis tubing according to the text protocol, use the dialysis tubing closure to clamp one end of the dialysis membrane and transfer the dialysis mixture into the tubing.

Then, clamp the open end and ensure that there are no leaks. Place the dialysis tube in a dialysis bucket with the pre-cooled Buffer A.Then, add a magnetic stir bar, ensuring that it will not touch the dialysis tubing while stirring. Dialyze the sample at four degrees Celsius with continuous stirring at 500 RPM and change the buffer at least four times over a period of 12 hours.

After the last buffer change, leave the sample to dialyze overnight. The following morning, remove the dialysis tube from the bucket and transfer its contents into a 500 mL beaker. Keep the protein solution on ice unless indicated otherwise.

Filter the protein solution through a 0.43 micron pore-sized membrane into a 500 mL glass bottle to remove any precipitated protein. After washing, charging, and equilibrating a chelating column according to the text protocol, adjust the refolded protein sample obtained to the composition of Buffer F by adding 25 mL of 5 M sodium chloride, 2.5 mL of 1 M tris-HCl, pH 8.0, and 2.5 mL of 2 M Imidazole stock solutions. Load the sample onto the column at a rate of 5 mL per minute or less and discard the flowthrough that contains the unbound proteins.

Then, wash the column with 50 to 100 mL of Buffer F to remove non-specifically bound proteins and discard the flowthrough. Elute the hexahistadine TIp3-LBD with 25 mL of Buffer G.Then, pool the flowthrough fractions containing the protein. After preparing Buffer H and dialysis tubing according to the text protocol, add hexahistadine tagged TEV protease to the hexahistadine tagged protein in the tubing.

Add a final molar ratio of one TEV to eight of protein. Place the clamped dialysis tube into the pre-cooled four L of Buffer H and incubate it at four degrees Celsius with continuous stirring for two hours. Then, change the buffer and continue dialysis overnight to allow the TEV-mediated cleavage reaction to complete.

After exchanging to Buffer I, filtering the protein solution and adjusting the sample again, load the sample onto a prepared five mL HiTrap chelating HP column. At a flow rate of five mL per minute, collect the flowthrough which contains the untagged TIp3-LBD. On cleaved protein, the cleaved hexahistadine tag and hexahistadine TEV are retained by the column.

Use five mL of Buffer F to wash the column to allow the untagged protein to flow through and collect the eluate. Then, after equilibrating a size exclusion column and concentrating and clarifying the protein sample according to the text protocol, load the sample onto the pre-equilibrated 26/60 gel filtration column. Apply Buffer A at a flowrate of four mL per minute and monitor the UV trace for protein elution.

TIp3-LBD elutes at a retention volume of 210 to 220 mL. Finally, following chromatography, pool the fractions then measure protein concentration and carry out SDS-PAGE. The protein isolation procedure demonstrated in this video yielded 10 to 20 mg of pure untagged TIp3-LBD per one L of bacterial culture.

As seen here, the protein eluted from the gel filtration column has a single, symmetrical peak, corresponded to a retention volume of 220 mL with a calculated molecular weight of 29 kDa. As shown here by SDS-PAGE, inclusion bodies contained predominately hexahistadine TIp3-LBD with an apparent molecular weight of 28 kDa, which is close to the value calculated from the amino acid sequence of 31.8 kDa. The hexahistadine tag removal, affinity chromatography, and gel filtration steps yielded highly pure protein.

CD spectroscopy using CDSSTR revealed that the secondary structure of hexahistidine TIp3-LBD was made up of 31%alpha-helix and 23%beta-sheet content. These were also close to the predicted values of 37%and 26%respectivelyr from sequence analysis using the JPred 3 server. This protocol requires a minimum of five days from start to end, and can be paused if necessary at three different stages.

When attempting this procedure, it is important to remember that thorough and complete resuspension of the inclusion bodies in the denaturing buffer by vortexing or pipetting up and down is critical for this whole realization. After watching this video, you should have a good understanding of how to refold and purify the ligand binding domain of the chemoreceptor TIp3. The purified protein can be used for binding acids to investigate the ligand's specificity for this receptor.

In addition, we have previously shown that the protein obtained by this procedure could be used to produce highly-ordered crystals, and that paved the way for the studies of the structural basis for its ligand specificity. This procedure might be generally useful for the production of mg amounts of chemoreceptors from other bacteria in a crystallizable and soluble form. We have used this protocol in its original or slightly modified form to refold and purify the ligand binding domain of other chemoreceptors of this type for crystallographic studies.

Please remember that in each separate case, optimization of the protocol, for example, the composition of the denaturing and refolding buffers and incubation times will likely be needed.