The overall goal of this procedure is to describe a yeast high throughput, two hybrid assay that reveals interactions between the Pregnan X receptor or PXR and the small molecule inhibitor ketoconazole. This is accomplished by first constructing the yeast two hybrid plasmids, one expressing the human P XR ligand binding domain and the other expressing the steroid receptor. COACTIVATOR one or SRC one presence of ketoconazole disrupts the interaction of human P XR with SRC one as revealed by liquid beta galacto assays.
Together with colormetric screening of colonies on a filter screening of PXR mutants in the presence of ketoconazole allows identification of mutations that render the receptor immune to the action of the drug, ultimately laxy expression by visual inspection and results of the liquid beta galacto. SASE assays are used to show changes in PXR activation in the presence of ketoconazole, which allows the identification of original residues involved in the interaction. We first had the idea for this method in Dr.Manny's lab where I'm an associate.
When we were confronted with incomplete structured data on PXR, the biochemical techniques would not allow us to perform with identification, especially for size of interaction that were very shallow and not readily amendable. To structure characterization. Based on the principle of detecting kiton resistant PXR mutants, we came up with an acid that will be tractable, feasible and could be performed within days in the very high throughput manner.
Though this method can provide inside into binding pharmacal force of small molecules on receptor proteins, it can also be applied to other systems such as any protein that has a pathogenic role in disease, particularly one limitations in structure biology approaches pre crude identification of bending residues to better understand and design small molecular target interactors. To begin amplify the human PXR ligand binding domain and full length human SRC one as described in the text protocol. Check PCR products by running on a 1%aros gel.
Next, purify the PCR products from the AROS gel. Digest the PCR products in two hybrid vectors as described in the text protocol and place the digestion samples in a 37 degree Celsius water bath for one hour. Purify the digested samples from a 1%agros gel, then ligate the digested and purified PCR products and two hybrid vectors before transforming into DH five alpha competent cells following overnight incubation at 37 degrees Celsius.
Pick the colonies and isolate the plasma DNA use restriction enzyme digestion to confirm the presence of the vector and insert, verify all positive plasma DNA by sequencing. Inoculate the yeast strain in five milliliters of YAPD and incubate overnight by constant agitation at 220 RPM and 30 degrees Celsius. The next morning inoculate the yeast one to 20 in five milliliters of YAPD and incubate to obtain an optical density at 600 nanometers of 0.2 by constant vegetation.
Pellet the cells at 2000 RPM in a micro centrifuge for two minutes and discard the supernatant. Then wash the pellet twice with autoclave water and once with 0.1 molar lithium acetate. After discarding the supernatant of the final wash, add the transformation reagents listed in the text protocol to the cell pellet and vortex to mix.
Then add the prepared PXR containing and SRC containing vectors before mixing. Again, pipette the transformation mixture into a polystyrene round bottom tube and agitate it for 30 minutes. Move the tube to a 42 degree Celsius water bath and incubate for an additional 30 minutes.
Next, centrifuge the tube at 2000 RPM for two minutes following removal of the supernatant. Wash the pellet once with autoclave water pipette 100 microliters of sterile water into the tube and resuspend the cells by gentle finger tapping and pipetting. Then plate the cells onto dropout medium without histidine and leucine and incubate at 30 degrees Celsius for three to four days to isolate transformants.
To begin the Xga filter assay, cut a triangle out of a nitrocellulose membrane to fit into a 10 millimeter portion of a Petri dish. Label the nitrocellulose membrane to mark the orientation. Place the pre-marked nitrocellulose membrane onto the yeast colonies ensuring that the membrane is in contact with the surface of the plate medium.
Remove the membrane. Place it colony side up on a three millimeter filter paper and place the membrane and the filter paper at minus 80 degrees Celsius for 15 minutes. Next, place two circles of three millimeter filter paper in a Petri dish and soak them with five milliliters of xal solution.
Drain excess buffer from the Petri dish and place the nitrocellulose membrane in the dish with the colony side up. Close the lid ensuring that the membrane is making complete contact with the filter paper and it is entirely wet. Wrap the Petri edition foil and incubate at 37 degrees Celsius for 30 minutes to overnight grow yeast and drop out liquid medium without histidine and leucine to an optical density at 600 nanometers of 0.7.
Transfer one milliliter of medium into a 1.5 milliliter micro centrifuge tube and centrifuge at 3000 RPM for two minutes. After discarding the supernatant, add one milliliter of Z buffer to the pellet and centrifuge. As before, add 150 microliters of Z buffer with tumor capto ethanol to resuspend the cell pellet.
Then add 50 microliters of chloroform and 20 microliters of 0.1%SDS vigorously. Vortex the sample for 15 seconds. Next at 700 microliters of ONPG solution, set the timer and incubate at 30 degrees Celsius long enough to allow the yellow color to develop.
After the color develops, note the total reaction time. Add 500 microliters of one molar sodium carbonate to stop the reaction. Then determine the absorbance at 420 nanometers.
As a final step, calculate Miller units as described in the text protocol. Since yeast has significant sterile production, laxy expression in yeast can be induced without the need for additional exogenous ligand. The xal lift assay revealed that laxy expression as visualized by the blue colonies is induced in the yeast strain transformed with PXR and SRC one.
This is shown in the beta glaces liquid assay as well. However, there is no induction of laxy expression in yeast transformed with empty vectors PXR alone or SRC one alone. Ketoconazole disrupts P XR and SRC one interactions in yeast since all the colonies from the replica filter were white, which was also shown by the significantly reduced beta galactic sase activity by liquid enzymatic assays.
When yeast transformations were performed with S rrc one on the P XR double mutant, it revealed that the colonies exposed to ketoconazole still retain laxy expression. This is similar to previous studies in this laboratory that showed the same PXR can be activated by a strong ligand, but is immune to the antagonistic effects of ketoconazole in mammalian assays. After watching this video, you should have a good understanding of how to perform a careful and where is could is to hybrid AC and the necessary modifications required for the study of small molecular bending residues on proteins.
Where attempting this procedure is important to remember to obtain a highly diverse library of mutants of your receptor, achieve good transformation efficiency of each 200 pluses, develop near identical replicas of the filter in place with or without drugs, and finally perform accurate visual and a chemical inspection for nase detection. Following this procedure, other methods like Ian 200 a Acids protein pro as acids and the iCal docking homologous models can be performed in order to answer additional questions like at least bend uc applicable to protein human cells. And if so, can model be made that defense, this residues as a pharmaco for.
