This method can help answer key questions related to the biophysics of mammalian ion channels including but not limited to the bestrophin family of ion channels. The main advantage of this technique is that it can be applied to other types of ion channels and generates key products for various downstream in vitro assays. The implications of this technique extend toward therapy of bestrophinopathies because assays using the purified protein contribute to our knowledge of how bestrophin channels work in the retina and how mutations in the BEST1 gene cause macular degeneration.
Twenty-four hours before viral infection, add an equal volume of trypan blue to a 15 microliter aliquot of HEK293F cell culture and check the cell density and viability using a hemocytometer under a microscope. After performing the cell count, plate 0.6 times 10 to the sixth cells per milliliter in a 500 milliliter volume in each of two disposable two liter flasks. Place the flasks into a 37 degree Celsius humidified incubator with 8%carbon dioxide.
Rotate the culture on an orbital platform at 135 rpm. On the day of infection, check the cell density and viability of a 15 microliter aliquot of the culture as before. A high cell viability of over 95%is essential for efficient infection and protein expression.
The expected cell density is about one times 10 to the sixth cells per milliliter and the expected total number of cells is about one times 10 to the nine. Add the P3 Baculovirus to the cells at a multiplicity of infection of five. To determine the amount of virus to inoculate, use the equation shown on screen.
Then incubate the inoculated cells for 24 hours. Twenty-four hours later, add sodium butyrate to a final concentration of 10 millimolar to each cell culture and return to the incubator set at either 37 degrees Celsius or 30 degrees Celsius for 48 hours. Forty-eight hours later, check the percentage and brightness of green cells which directly represent the protein under a fluorescence microscope.
Harvest the cells by centrifuging at 1, 000 times g at four degrees Celsius for 20 minutes. Remove the supernatant and resuspend the cell pellets with phosphate buffered saline to a final volume of approximately 80 milliliters. Split each cell suspension between two 50 milliliter conical tubes and centrifuge for 20 minutes at 1, 000 times g and four degrees Celsius.
To begin the protein purification procedure, first thaw the cell pellets in a stirring water bath at room temperature. When the pellets are thawed after 10 to 15 minutes, resuspend the cells in twice the volume of buffer A supplemented with proteinase inhibitors. Pipette up and down extensively to obtain a homogenous cell suspension.
Lyse the cells using a high-pressure homogenizer. Run the cell suspension through the homogenizer at seven to 10 megapascals three to four times to achieve complete homogenization. Keep the cell lysate on ice for two to three minutes between rounds.
Add detergent to the cell lysate. Incubate with agitation for one hour at 20 degrees Celsius to extract the membrane proteins. Following the incubation, centrifuge the cell lysate at 150, 000 times g in an ultra centrifuge at four degrees Celsius for one hour.
After centrifugation, the clear cell lysate will be sandwiched between the pellet at the bottom and a cloudy layer on top. Use a 10 milliliter transfer pipette to carefully collect the clear lysate and then switch to a one milliliter pipette for the last few milliliters of lysate. Apply the lysate to a five milliliter HisTrap nickel NTA affinity column pre-equilibrated with buffer A.Next, wash the column with 25 milliliters of buffer B followed by 40 milliliters of buffer C.The protocol can be paused here.
The protein will remain stable while attached to the column overnight. After washing, attach the column to a fast protein liquid chromatography system and dilute the protein from the column with 13 milliliters of buffer D with fractionation. Collect the protein-enriched fractions according to the UV absorbance readout.
Measure the eluted protein product concentration on a microvolume spectrophotometer by reading the absorbance at 280 nanometers. To remove the GFP10x his-tag, add the TEV protease at a one-to-one mass ratio and incubate at four degrees Celsius for 30 minutes. Concentrate the protein into a final volume of 400 to 500 microliters with a 15 milliliter centrifugal filter unit by spinning at 4, 000 times g in a four degree Celsius centrifuge for variable intervals beginning with 10 minutes.
Transfer the concentrated protein to a clean 1.5 milliliter tube and remove any precipitate or bubbles from the concentrated product. Spin at 12, 000 times g for five to 10 minutes at four degrees Celsius, then transfer the supernatant to a new 1.5 milliliter tube. Use a one milliliter syringe and a round tip needle to load the final product on an FPLC system for size exclusion chromatography with a size exclusion column pre-equilibrated with buffer E.Well-behaved proteins run as a single peak.
Collect the protein fraction or multiple fractions that correspond to that peak. Concentrate the protein with a four milliliter or 0.5 milliliter centrifugal filter unit of the same molecular weight cutoff as previously used. Spin at 4, 000 times g at four degrees Celsius to a final concentration of five to 10 micrograms per microliter.
Use the spectrophotometer to check the final product concentration. The fluorescence intensity observed in transiently transfected adhesive HEK293 cells seen here is a good indicator for the projected protein expression level in suspended HEK293F cells shown here. A successful purification is indicated by a single main peak at the expected dilution volume in size exclusion chromatography and a single dominant band on a denatured SDS page gel.
While attempting this procedure, it's important to remember to keep everything on ice during purification and if performing multiple purifications at the same time, change gloves frequently and make sure not to contaminate samples. Following this procedure, other methods like x-ray crystallography and lipid bilayer assays can be performed to answer important questions like structure function relationships. After its development, this technique paved the way for researchers in the field of inherited retinal diseases to explore the causes of bestrophinopathies at a molecular level.
