Biochemistry
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Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy
Chapters
Summary January 7th, 2019
This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.
Transcript
This protocol enabled us to solve the structure of human TRPC3, a member of an important, but understudied family of ion channels. The main advantage of this technique is the high efficiency with which it can be applied to express and purify a variety of proteins, for both structural and functional studies. This method can provide an efficient protein expression and purification system, for use in studying protein biochemistry and biophysics and can be applied to a wide variety of ion channels and other membrane proteins.
First, check the relative virus expression by viewing the GFP fluorescence of the virus in a sample of the virus producing culture. In a baffled bottom Erlenmeyer culture flask of sufficient size, prepare a desirable volume of a HEK293 mammalian cell suspension culture, at a concentration of 3.5 to 3.8 million cells per milliliter in the expression medium, supplemented with 1%sterile FBS. Add 8%by volume of prepared P2 virus stock solution and incubate in an Orbital Shaker at 37 degrees Celsius and 135 RPM.
At 12 to 18 hours post-infection add 10 millimolar sodium butyrate, incubate at 30 degrees Celsius for the amount of time necessary for optimal protein expression. After this, centrifuge at 2, 880 times gravity for 20 minutes to harvest the cells. Wash and resuspend the cells in approximately 100 milliliters of TBS per liter of cells harvested.
Centrifuge again at 2, 880 times gravity for 20 minutes and collect the cell pellet. Also collect small, one milliliter cell pellet harvests at varying time points and solubilize for two hours at four degrees Celsius with agitation in the presence of different detergents and/or additives. Clarify these small whole cell solubilized samples by ultracentrifugation at 235, 000 times gravity and at four degrees Celsius for 10 minutes.
Then, run them as 30 microliter samples on a SEC Chromatography column, to determine the best time for expression and the best solubilization conditions. Thaw the pellet in 100 milliliters of buffer per liter of cells harvested. Once the cells are thawed, pipette or stir them to ensure the solution is homogeneous.
Let the cells solubilize at four degrees Celsius in a beaker immersed in ice for two hours while being stirred by a stir bar. Next, remove any cell debris by ultracentrifugation at 235, 000 times gravity and at four degrees Celsius for one hour. Run a 30 microliter sample of the supernatant on an SCC column by HPLC to verify the protein quantity and to visualize the target protein by GFB signal output.
Apply the cobalt affinity resin bound supernatant containing the solubilized protein to a gravity column and collect the flow-through. Run a 30 microliter sample of the flow-through on a SCC column to verify the protein is binding to the resin. Then, wash the resin with 10 column volumes of buffer.
Run a 30 microliter sample of the wash on a SCC column to check for protein loss. Using buffer, elute the resin bound human TRPC3. Run a 90 microliter sample of the eluant that has been diluted one to 100 on an SSC column to check that the protein has been eluted and to verify the presence of a GFP signal at the position corresponding to the target protein size.
Add Thrombin at a one to 20 molar ratio and add 10 millimolars of EDTA to the eluted sample. Incubate at four degrees Celsius for three hours. After this, transfer the eluant to a 15 milliliter centrifugal filter tube.
Spin the tube at 2, 800 times gravity and at four degrees Celsius in five minute increments to concentrate the eluant to 500 microliters or less. Pipette the protein solution up and down between spins to resuspend the protein and prevent over concentrating. Load the concentrate onto an SSC column in buffer.
Run fast protein liquid chromatography and collect 300 microliter fractions. Then, combine the peak fractions that contain the TRPC3 tetramer as visualized by the UV absorbance signal. And concentrate again to a final concentration of at least five milligrams per milliliter.
The human TRPC3 is whole cell solubilized in a variety of detergents with a critical micelle concentration of 0.01 to 20 micromolars, and was run in a DDM/CHS containing buffer. Although DDM/CHS shows the highest solubility of human TRPC3, The peak position appears too large to be the tetrameric human TRPC3. After whole cells solubilization the cell lysis debris is removed and the solubilized protein is loaded on an SSC column and run on HPLC in DDM/CHS detergent containing buffer, to compare the absolute solubility and peak volume of human TRPC3 under different conditions relative to a TRPM4 control.
All of the different detergent solubilized TRPC3 samples show peak positions around 11.9 milliliters which is likely too large because the tetrameric form of human TRPC3 has a smaller molecular weight than the positive human control TRPM4. Two different solubilization and running buffers containing DDM/CHS and digitonin are then tested. The protein run in buffer containing digitonin yields the highest peak in a reasonable position relative to the positive control.
While a small scale purification using 25 milliliters of cells shows several broad peaks, the protein shows features of a single tetrameric human TRPC3 channel in 2D classification by negative-stain. Additives are then screened based on the physiological character of human TRPC3. EDTA shows a remarkable affect on stabilizing human TRPC3 in a intact tetrameric peak position and significantly increased the number of particles in the cryo-EM micrograph and decreased the noise in the background.
The quality of the final results depends on good troubleshooting of the FSCC profiles on HPLC. And on completing all of the purification steps quickly, ideally in a single day. Structural determination, antibody generation, and functional studies such as, binding assays or electrophysiology can be performed after this procedure.
This technique can and has been adapted to study other ion channels and protein families and to produce purified proteins for applications beyond structural determination. The appropriate precautions for managing cell culture biohazards such as working in a laminar flow hood, wearing protective clothing, and properly disposing of waste, should be taken.
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