The overall goal of this procedure is to generate a transporter that is sufficiently stable for structural studies, and use this modified transporter to produce crystals which defract to high resolution by x-ray crystallography. This method can answer key questions in the field of structural biology, such as how to solve the structure of membrane proteins which are important drug targets. The main advantage of this technique is that it can be used to select from a drug-bound conformation using a functional assay.
Though this method can provide insight into neurotransmitter transporters, it can also be applied to receptors, channels, and soluble proteins, which bind small molecules with high affinity. Thoroughly resuspend trypsonized HEK293S GnTI minus cells in DMEM supplemented with 10%fetal bovine serum to a density of 0.5 million cells per milliliter in a disposable pipette reservoir. Using a multi-channel pipette, add 100 microliters of cells to each well of a poly-D-lysine coated plate.
Resuspend the cells in the pipette reservoir after filling each plate to ensure an even distribution of cells. Incubate the cells in the 37 degree Celsius incubator with 8%carbon dioxide. Replace the cell media one hour prior to transvection.
Prepare the DNA transvection reagent complexes by mixing 450 nanograms of DNA with 45 microliters of serum-free DMEM for each construct in the cert TC background to be screened. Then add 1.6 microliters of the transvection reagent to 45 microliters of serum-free DMEM and mix. Immediately add the diluted transvection reagent solution to the DNA solution and mix.
Do not mix solutions in the reverse order. Wait 10 to 15 minutes before adding 20 microliters of the transvection reagent DNA mixture to four of the wells. Incubate the cells with 200 nanomolar citalopram and 25 microliters of TBS for five minutes at room temperature.
To solubolize the cells, add 25 microliters of eight millimolar C12M, one millimolar CHS, and protease inhibitor cocktail in TBS. Incubate the cells for one hour at room temperature. Then add 50 microliters of TBS with 20 nanomolar tritiated citalopram, 0.1%bovine serum albumin and milligrams per milliliter of his tag affinity scintillation proximity assay or SPA Beads, to three wells for each construct.
For the last well add the same TBS solution, but supplement with 100 micromolar sertraline to determine non-specific binding. Ensure that the his tag affinity SPA Beads are thoroughly mixed when adding them to the 96 well plate. Measure tritiated citalopram binding using a 96 well scintillation counter at room temperature with a one minute count time per well.
Continue counting plates until total counts plateau at approximately 36 hours. Following measurement, heat the plates for 15 minutes in a heating block with a heated lid at 33 degrees Celsius. Measure tritiated citalopram binding again after heating.
Repeat these steps with modification of the heating step. Adjust the heating temperatures depending on the apparent melting temperature of the target protein and the thermostability of the most stable construct. Continue to heat plates until all constructs have low specific counts.
Grow HEK293S GnTI minus cells in suspension at 37 degrees Celsius with 8%carbon dioxide and 85%humidity on a shaker at 130 RPM and 293 expression media supplemented with 2%fetal bovine serum. Infect 10 liters of the cells with the P2 virus at a multiplicity of infection of two and a density of three million cells per milliliter. 12 to 16 hours post-infection, add sodium butyrate to a concentration of 10 millimolar from a one molar stock.
48 to 60 hours post-infection, harvest the cells by centrifugation at 4, 000 times G for 15 minutes. Remove the supernatant. Resuspend cells in 150 milliliters of TBS with 2 micromolar escitalopram or other SERT inhibitors.
the cells from 10 liters of culture in warm water approximately 30 degrees Celsius and resuspend them by rapidly passing the cells through a 10 milliliter pipette until homogeneous. Add all of the cells to a beaker with a stir bar and add all of the detergent solution to the cells while stirring. Solubilize the cells at four degrees Celsius for one hour with stirring.
Spin the lysate at 8, 000 times G for 15 minutes at four degrees Celsius. Decant the supernatant into clean ultra centrifuge tubes and discard the pellet. Then spin the supernatant at 100, 000 times G for one hour in an ultra centrifuge.
Following ultra-centrifugation, filter the supernatant through a 0.2 micron filter and discard the pellet. Next pass the lysate over 10 milliliters of strep affinity resin packing into a column using a perisaltic pump equilibrated in wash buffer. Then connect a strep affinity column to a fast protein liquid chromatography system and wash the column at two milliliters per minute with 66 milliliters of wash buffer.
Elute the purified protein in the same wash buffer supplemented with five millimolar desthiobiotin at 0.5 milliliters per minute using 33 milliliters of buffer. Collect one milliliter fractions. The peak fractions will be approximately 10 milliliters.
To form the transporter antibody complexes, first digest the purified protein overnight at room temperature with thrombin to remove tags and Endo H for deglycosylation. Concentrate the protein to a concentration of 10 milligrams per milliliter and a volume of 250 to 300 microliters using a 100 kilodalton molecular weight cutoff centrifuge protein concentrator. Mix the concentrated SERT protein with 8B6 Fab at a molar ration of one to 1.2 in a volume less than 500 microliters.
Centrifuge the mixture at 100, 000 times G and four degrees Celsius for 20 minutes. Following centrifugation, collect the supernatant containing the SERT Fab complex, and discard the pellet. Separate the complex using fast protein liquid chromatography on a size exclusion column.
Equilibrate it with supplemented TBS at 0.5 milliliters per minute. Prior to crystallization, concentrate the peak fractions from the separation of the complex to two milligrams per milliliter using a 100 kilodalton molecular weight cutoff centrifuge protein concentrator. An absorbance of two AU at 280 nanometers is equal to one milligram per milliliter.
Add additional Fab at a ratio of one to 0.05 complex to free Fab. Also add 10 micromolar free S citalopram. After centrifuging the sample, collect the supernatant containing the SERT Fab complex and discard the pellet.
Set up a hanging drop 24 well screen at four degrees Celsius according to table one in the text protocol. Pipette 500 microliters of each reservoir solution in a low profile 24 well plate with sealant applied to each well. Pipette 1.5, 1.75, and two microliters of the SERT Fab complex onto an 18 millimeter siliconized glass cover slip.
Then pipette one microliter of reservoir solution on top of the protein sample. Single crystals will appear within approximately three days and grow to 100 to 175 micrometers after 14 days. Shown here are representative results for screening thermostability in the presence of tritiated citalopram.
The melting temperature values plotted against maximum binding show mutants with high thermostability and expression. Displayed here are the thermostability curves for wild-type SERT TC and the top three mutants, Y110A, I291A, and T439S. This microscopy image shows HEK293S GnTI minus cells expressing SERT CC with green fluorescence present on the cell membrane.
Fluorescence detection size exclusion chromatography of SERT CC shows a major peak at 15 milliliters. Analysis by SDS-PAGE displays a single protein species largely free of contaminants. Representative separation of transporter antibody complexes by size exclusion chromatography is shown.
The major peak eluding at 11.5 milliliters contained both SERT and Fab as shown on an SDS-PAGE gel. Light microscopy of the SERT antibody complex bound to S citalopram after two weeks of growth shows prism shaped crystals of approximately 100 to 175 microns. After watching this video you should have a good understanding of how to identify mutations that stabilize a membrane protein in a ligand-bound confirmation and how to set up crystallization screens with protein antibody complexes.
When attempting to do this procedure it is important to remember that ligands are necessary for the stabilization of SERT TC and are essential for crystallization of SERT CC.The implications of this technique extend toward therapy of psychiatric conditions because SERT is the molecular target of many antidepressant drugs. Following this procedure, the function of purified SERT can be characterized using biochemical and biophysical methods.
