The aim of this procedure is to purify the amyloid precursor protein intracellular domain, also known as A ICD, and to present a protocol for thermal aggregation and characterization of the A ICD by atomic force microscopy. This is accomplished by first expressing A GST fusion of A ICD in bacteria. Next, the G-S-T-A-I-C-D protein is purified cleaved with thrombin to separate the GST from A ICD and the GST is removed.
Then the thermal aggregation of A ICD is induced. Finally, the A ICD aggregates are imaged using atomic force microscopy. Ultimately, results can be obtained that show that the A ICD can be purified to homogeneity in quantity sufficient for biochemical and biophysical analysis.
The main advantage of this technique over existing methods, which require complex chromatography steps is that highly pure and soluble. A ICD can be isolated on the benchtop using standard reagents and equipment. A ICD produced by this protocol is suitable for further biochemical and biophysical characterization.
Demonstrating the procedure will be Amina ti. A former postdoc for my laboratory, Dr.Andres Oberer, will demonstrate how to perform the atomic force microscopy imaging of A ICD To express the recombinant A PP intracellular domain or A ICD. Begin with BL 21 cells transformed with the pgx 41 vector, harboring the human A ICD fused to the C terminus of GST with a single colony inoculate 10 milliliters of LB broth with ampicillin and incubate overnight at 37 degrees Celsius with vigorous shaking the following morning.
Incubate 400 milliliters of LB ampicillin in a one liter flask with five milliliters of the overnight culture. Incubate at 37 degrees Celsius with vigorous shaking until the optical density at 600 nanometers has reached 0.4 to 0.6. This will normally take two to 2.5 hours induce expression of G-S-T-A-I-C-D.
We're adding 0.4 millimolar of IPTG and incubated 37 degrees Celsius with vigorous shaking for five hours. Centrifuge the cells at 6, 000 times G for 15 minutes of four degrees Celsius and store the pellet at minus 80 degrees Celsius. After preparing lysis, buffer and throwing bacteria on ice, add 20 milliliters of buffer to the pellet and using a combination of vortexing and tation on ice with a pipette thoroughly.
Reese suspend the bacterial pellet pass the Reese suspended bacterial lysate through a pre chilled emulsifier or French press. At 30, 000 PSI pass the lysate through the emulsifier a second time and collect the lysate in a pre chilled 50 milliliter tube. Centrifuge the lysate of 15, 000 times G and four degrees Celsius for 30 minutes.
Separate and transfer the supinate into a pre chilled 50 milliliter tube. Add 500 microliters of a 50%slurry of glutathione arose. Rotate for 30 minutes at four degrees Celsius while the sample is rotating.
Make fresh elution buffer after the incubation. Pour the lysate and slurry into a disposable five inch polystyrene chromatography column with course filters. Wash the column five times with three milliliters of PBS.
Cap the bottom of the column and add 500 microliters of room temperature, elution buffer. Cap the top of the column and rotate for five minutes of room temperature. Collect the early weight into a chilled 1.5 milliliter micro refuse tube.
Repeat the elution two more times. Dialyze the eluate in a 10, 000 molecular weight cutoff dialysis membrane in four liters of PBS overnight of four degrees Celsius the following day. Dialyze against an additional four liters of PBS at four degrees Celsius for one hour.
Remove the protein from the dialysis cassette and perform a protein quantification assay to determine the yield of G-S-T-A-I-C-D. A typical purification from 400 milliliters of culture will result in yields of greater than 20 milligrams of purified protein are concentrations of 10 to 20 milligrams per milliliter. Aliquot the protein into 200 microliter, aliquots, and freeze of minus 80 degrees Celsius.
To liberate the A ICD from GST thaw, 200 microliters of purified G-S-T-A-I-C-D and add 20 micro releases of one unit per microliter of thrombin incubate overnight at 37 degrees Celsius the following day, clear any aggregated material by centrifugation at 20, 000 times G for 30 minutes of four degrees Celsius. Transfer the SUPINATE into a pre chilled micro tube to remove GST and thrombin. Add 50 microliters of 50%glutathione aro slurry and 50 microliters, 50%p and wino zaine aro slurry respectively.
Incubate with rotation for five minutes of room temperature. Centrifuge briefly to sediment the agro beads and remove the supinate into a fresh tube. Extract the GST four more times with glutathione.
Aros perform a protein quantitation assay on five microliters of the purified A ICD. Typical yields from a 400 milliliter culture are 50 to 100 micrograms at a concentration of 0.2 to 0.5 milligrams per milliliter. After diluting freshly prepared A ICD in PBS to a concentration of 0.1 milligram per milliliter induced thermal aggregation by incubating the samples at 43 degrees Celsius at 800 RPM in a heated shaker for 48 hours.
Dilute the sample 20 fold with deionized water and spot two microliters onto freshly cleaved. Mica dry the sample in a desiccate overnight to visualize the aggregated A ICD use atomic force microscopy in tapping mode with gold goated. Micro sharpp nitride cantilevers with a nominal tip radius of two nanometers.
Typical tapping amplitudes during imaging, a 10 to 20 nanometers at the resonance frequency of the cantilevers. The expression and relative enrichment of G-S-T-A-I-C-D is shown here. After lysis, the majority of the fusion protein is present in the soluble fraction, and therefore extraction from inclusion bodies is not required.
The material alluded from the glutathione arose column is greater than 95%pure. In the preparation shown here, the concentration of protein alluded from the column was 14.5 milligrams per milliliter, and the yield was 22 milligrams. Cleavage of 200 microliters of this preparation overnight with 20 units of thrombin resulted in almost 100%cleavage of the fusion protein.
The amount of thrombin used is low enough as to not be detectable by kumasi. Blue staining, removal of the thrombin and GST resulted in some loss of material. However, it was greater than 90%pure with a yield in this prep of 70 micrograms of A ICD at a concentration of 0.35 milligrams per milliliter.
A ICD aggregates, imaged by a FM are typically steroid amorphous and range in size from 50 to 100 nanometers. Aggregated material is not detectable when an equimolar amount of the chaperone UBI Quillin one is added to the aggregation reaction. After watching this video, you should have a good understanding on how to purify EICD induce its aggregation and image aggregate bay A-F-M-E-I-C-D.
Purified by this technique can be applied to other methods such as light scattering and filter trap assays to answer questions regarding the effect of molecular chaperones on EACD aggregation.
