The overall goal of this procedure is to assemble A PDZ dependent cystic fibrosis transmembrane conductance regulator, or CFTR macromolecular signaling complex. In vitro to do this, the recombinant tagged fusion proteins are expressed in bacteria and purified. Next cell lysates are obtained from cultures that express CFTR or CFTR binding proteins.
Then the CFTR and binding proteins are combined in vitro to assemble the CFTR containing macromolecular signaling complex. Western blotting is then performed, which demonstrates a formation of CFTR macromolecular complexes and is then assessed via western blotting. The main advantage of this technique over existing methods is that we can directly detect and visualize the formation of a C CT R containing high-protein macromolecular complex individual.
So this method can provide insight into the formation of CT R containing macromolecular complex. It can also be applied in assembling non CFTR involved non-protein complex such as sex C two macromolecular signaling complex. Most recently, using this same method, we have demonstrated that our chemo receptor CXCR two physically formed protein complex with the downstream effect.
PRC beta three mediate the wild nerve one in neutrophils Using PCR. Amplify the DNA that encodes the last 50 to 100 amino acids containing the PDZ motifs at C terminus. For the CAMP transporter, MRP four and the PDZ one domain of sodium hydrogen exchanger regulatory factor or N-H-E-R-F.
Next, clone the RP four PCR R products into PGE X four T one to generate GST fusion proteins and the PDZ one domain of N-H-E-R-F into PET 30. To generate hiss s fusion proteins, transform the bacteria by heat shock. In SOC medium, in a protease deficient e coli strain, grow the culture overnight at 37 degrees Celsius in a shaking incubator in 50 milliliters LB medium containing appropriate antibiotics for the vector.
The next day dilute the culture one to 10 and 500 milliliters. Luria berti medium, then incubate the culture again shaking at 37 degrees Celsius after two hours. Induced transcription of the recombinant protein with 0.5 to one millimolar IPTG for four hours at 30 degrees Celsius.
After the incubation, transfer the cultures to 250 milliliter centrifuge tubes and pellet the cells by centrifugation at 8, 000 times G for 10 minutes at four degrees Celsius. Following the spin, discard the supernatant and add 20 milliliters of sucrose buffer containing lysozyme. One milligram per milliliter 0.2%Triton X 100 and protease inhibitors to lys the cells, transfer the bacterial suspension into a clean 50 milliliter centrifuge tube.
Mix on a rotary shaker for 30 minutes at four degrees Celsius. Then spin at 20, 000 times G for 30 minutes at four degrees Celsius. After the centrifugation, the supernatant will be clear and an insoluble app pellet will be present at the bottom of the tube.
Collect the clear supernatant into a new 50 milliliter conical tube. Then to the supernatant, add one milliliter of the appropriate resin. Aero beads suspended as a 50%slurry in sucrose buffer glutathione.
Aero beads should be used for GST fusion proteins and talon beads for hiss S fusion proteins. Mix for four hours at four degrees Celsius on a rotary shaker. Then spin the beads at 800 times G for two minutes.
Then wash them by resus, suspending them in 15 milliliters of PBS and mix them on a rotator for two minutes. And finally discard the supernatant. Repeat the step for six times after the sixth wash.
To elute the protein from the beads, add two milliliters of the appropriate elution buffer for every one milliliter of beads and mix for 10 minutes at room temperature. Pour the whole eluded protein with beads into a column and collect the flow through as eluded protein. Next, transfer the UD protein to dialysis tubing.
Dialyze the eluted proteins against two liters of PBS while stirring at four degrees Celsius. Change the PBS every four hours for four times following dialysis. Concentrate the protein to 0.5 to one milliliters.
Using a 10, 000 megawatt cutoff cent con filter. Aliquot the sample into 200 microliters each. Save one for analysis and place the others at minus 80 degrees Celsius for storage.
Determine the protein concentration by the Bradford Method. Assess protein quality by SDS page using BSAS standards. If the integrity of the protein is not satisfactory, secondary purification procedures such as gel filtration or ion exchange may be used.
HEK 2 93 cells expressing wild type flag CFTR should be cultured as described in the accompanying document. Remove the medium from the cells and wash with PBS. Then to lys the cells, add 500 microliters of lysis buffer for each 60 millimeter culture dish and rock the cell lysates for 20 minutes at four degrees Celsius.
Transfer the lysates to new micro centrifuge tubes. Then to remove the insoluble proteins centrifuge at 16, 000 times G for 15 minutes at four degrees Celsius and collect supernatants. Determine the protein concentration by the Bradford assay to assemble the CFTR containing macromolecular complexes in vitro.
Begin by combining 20 micrograms of purified G-S-T-M-R-P four C terminal 50 amino acid fusion protein and increasing amounts ranging from zero to 40 micrograms of purified hiss S PD zk one fusion protein in 200 microliters of lysis buffer and a 1.5 milliliter micro centrifuge tube. Mix the two proteins on a rotary mixer at 22 degrees Celsius for one to two hours. Next, add 20 microliters of a 50%slurry of glutathione beads into the protein mixture and continue to mix for another one hour.
This step is also referred to as pairwise binding in which G-S-T-M-R-P four CT 50 binds to SPD zk one. During this time prepare the HEK 2 93 cell lysates that over express wild type flag CFTR as described in the previous section of the video following the pairwise binding step. Wash the complex twice with lysis buffer span at 800 times G for one minute for each wash and carefully aspirate the supernatant after each wash.
Use caution not to aspirate the beads from the bottom. Add the HT K 2 93 cell lysates to the beads and gently mix on a rotary mixer at four degree Celsius for three hours or overnight after the incubation. Wash the beads three times with lysis buffer centrifusion after each wash as before, after the last wash.
To elute the proteins add 30 microliters of five x sample buffer and incubate in 37 degrees Celsius water bath for 10 to 15 minutes. Then spin at 5, 000 times G for one minute to precipitate the beads. Next, load all the Eliot onto a four to 15%SDS page gel and run it at 200 volts to the end of the gel following the run Transfer protein bands to the PVDF membrane for 1.5 hours at 60 volts on a BioRad transfer apparatus.
After the transfer is complete, block the membrane in TBS tween containing 5%non-fat milk for one hour. Proceed with western blotting as described in the accompanying document to visualize CFTR interacting proteins. A-C-F-T-R containing macromolecular signaling complex was assembled in vitro as described in this video as shown in this pictorial representation.
A macromolecular complex formed between the C terminal 50 amino acids of Mr.RRP four PD zk one and full length CFTR to determine the dose dependency of the macromolecular complex formation. Increasing amounts of the intermediary protein PD ZK one were included as shown here. Complex increased dose dependently suggesting that the intermediary protein PD ZK one mediated the interaction between CCF TR and Mr.Rrp four.
While attempting this procedure, it's important to remember not to boil the the FTR container micro molecule complex samples prepared for western blotting as the FTR aggregation is a common problem. Instead, the samples should be heated at 37 degree for 10 to 15 minutes before loaded in the gel Following this procedure. Other methods like coun precipitation can be performed in order to assess the endogenous CFTR complexes in cells such as airway, epithelial cells, and the gut epithelial cells.
After watching this video, you should have a good understanding of how to assemble A PTC dependent CFDR Macromolecular Signaling Complex in vitro.
