This video will demonstrate a method to set up high throughput crystallization trials of purified membrane proteins using Lipic B cells. First B cells are prepared by mixing a lipid and a detergent purified membrane protein is incorporated into the B cell mixture and crystallization trials are performed using a nanoliter robot visual inspection. Using a standard light microscope reveals the presence of crystals.
Once formed, the protein crystals can be extracted and frozen for data collection and structured determination. The main advantage of this technique is that owing to the unique phase behavior of B cells, standard equipment and robotics available in the lab can be utilized for crystallization. Setup B cells is such a simple technique and allows you to take your target membrane protein out of the detergent my cell, and place it into a lipic medium.
This gives you an entirely new set of conditions to screen. It is such a simple method that has been successfully used on many membrane proteins that there's just no reason not to try it. The first step in B cell based crystallization is the preparation of a B cell forming lipid amphi mixture.
This process requires considerable effort and is the most time consuming. Note that it may take several hours to complete. B cells can form in a variety of lipid amphi combinations and over a wide range of concentrations.
For suggested starting points, see table one in the accompanying written protocol begin by preparing one milliliter of 35%DMPC capso mixture at a 2.8 to one molar ratio. To do this, weigh out 0.26 grams of DMPC and 0.09 grams of Chapo in a tube. Bring the volume up to 1.0 milliliters using deionized water vortex the mixture.
Next, warm the mixture to about 40 degrees Celsius in a water bath until it becomes gel-like thereafter. Place the tube on ice. The sample should become fluid like again.
Then vortex again for a few minutes, continue cycling through the warming, cooling and vortexing steps until the lipid is completely dissolved. Note that as more cycles are performed, the mixture may become cloudy upon cooling upon completion. The mixture will be a clear gel at or above room temperature, and a viscous liquid on ice by cells can be placed at minus 20 degrees Celsius for long-term storage.
Next purified proteins are incorporated into the B cell medium. This is a simple process and should be performed on the same day as crystallization, which is shown in the next section of the video. Thaw the DMPC capso B cell mixture at room temperature until the phase changes to a clear gel.
Place the mixture on ice to liquefy it and then briefly vortex it to reestablish a homogeneous B cell phase. From this point on, keep the B cell mixture and purified protein on ice. This will keep the B cell in a liquid phase, making it amenable to pipetting.
Next, add the B cell mixture to the purified detergent solubilized protein in a ratio of one to four, volume to volume. Then gently pipette up and down until the solution becomes clear and homogeneous. If bubbles appear, briefly, spin them down in a tabletop centrifuge.
Incubate the mixture on ice for at least 30 minutes to promote complete incorporation of the protein into by cells. The protein by cell mixture is now ready for crystallization trials. Crystallization trials will be demonstrated using a mosquito crystallization robot.
Cool, a 96 well V bottom plate by placing it on ice. Pipette the protein by cell mixture into the plate to ensure that the viscosity of the solution is minimal. Keep the protein by cell mixture on ice until the final step.
The robot used here has five platforms. Place the microplate containing the reservoir on platform three and the crystallization lid onto which the drops will be dispensed on platform five. Then place the protein by cell plate on the platform four closest to the dispensing position.
This assures that the protein by cell mixture is the last to be picked up by the robot and it is immediately released. Using the software initiate the run, the robot will pick up the reservoir solution followed by the protein by cell mixture and dispense them onto the lid as drops to prevent heating and increased viscosity during dispensing. Do not program the mosquito to mix the reservoir with the protein by cell mixture as soon as the run is complete.
Remove the buy cell protein plate from the instrument and place it on ice. Remove the lid from the instrument and place it on the plate containing the reservoir solution such that the drops are inverted. Incubate the plate at 20 degrees Celsius.
Note that other temperatures may also be tested for crystal formation and optimization. Higher temperatures induce the lamellar phase, which has the advantage of preor organizing the protein in layers. Temperatures between four degrees Celsius and 20 degrees Celsius can be screened.
Temperatures below four degrees Celsius may cause the lipids to precipitate over extended periods of time on the first, third day, and weekly thereafter, assess crystal appearance and growth using a standard light microscope. Shown here are a brightfield image and a UV image of needle shaped crystals observed in assault only condition. Fluorescence was not detected from the crystals indicating a false positive in this image.
A rod-shaped crystal formed when methyl propane diol was used as a precipitant. The crystal fluoresce weekly indicating that it might be a protein crystal, but was found to be non-pro tenacious using x-ray diffraction. Shown here is a crystal observed about four weeks after setting up trials.
The strong fluorescence under UV light confirms it is a protein crystal. As you watched on this video. Once B cells are available, they can be mixed directly with purified protein and from this point on crystallization proceeds almost exactly as standard detergent based protocols.
Another distinct advantage is the ability to dope B cells with specific lipids for optimization purposes. There's really a lot of versatility with the technique and it is so simple to use that it should be tried for all membrane protein crystallization projects.
