The overall goal of this procedure is to demonstrate how to use zinc finger domains to deliver functional proteins such as green fluorescent protein into mammalian cells. This is accomplished by first generating an expression plasmid containing the emerald green fluorescent protein MGFP translationally fused to zinc finger domains. The second step is to express the fluorescent fusion protein in bacteria.
Next, the bacterial cells are lysed and the fluorescent fusion protein is purified. The final step is to incubate mammalian cells with the purified fluorescent fusion protein to allow uptake. Ultimately, the fluorescence of treated cells can be quantified by flow cytometry to determine the efficiency of fusion protein delivery into the cells.
The main advantage of thief or other protein transduction domain like HIV derived tight peptide or poly arginine, is that it can facilitate high level of cytosolic delivery without compromising the activity of field enzymatic cargo. To begin PCR, amplify the emerald GFP or MGFP sequence from a plasmid According to the text protocol, purify the PCR product using a gel extraction kit and then quantify the DNA concentration using a spectrophotometer. Next, obtain a PET 28 expression vector containing the sequence of alanine.
Substituted two fingers, zinc finger or two FZ domains, digestive vector and the MG F-P-P-C-R product with the XMA one and SAC one restriction enzymes using 10 units of enzyme per microgram of DNA and incubate the digests for three hours at 37 degrees Celsius. Purify the DNA fragments by gel extraction and measure the concentrations of the vector and the MG F-P-P-C-R product. Using a spectrophotometer, combine the MG F-P-P-C-R product and 50 to 100 nanograms of the vector in a single tube at a six to one insert to vector molar ratio.
Then add one units of T four DNA ligase and incubate the reaction at one hour at room temperature to transform bacteria with the ligated plasmid.Thaw. A 50 microliter aliquot of chemically competent e coli cells on ice. Then gently mix the cells with 100 to 20 nanograms of the ligated DNA and incubate the cells with the DNA on ice for 30 minutes.
After the incubation, heat shock the cells at 42 degrees Celsius for 90 seconds. Add two milliliters of SOC medium to the tube and allow the cells to recover for one hour at 37 degrees Celsius with shaking to select for transformants. Spread 100 microliters of the culture on LB agar plates containing 50 micrograms per milliliter of can mycin and incubate the plates overnight at 37 degrees Celsius the following day.
Inoculate a six milliliter culture of LB containing 50 micrograms per milliliter of can mycin with an isolated colony from the overnight plate. Incubate the culture overnight at 37 degrees Celsius with shaking. The next day, collect and mini prep the cells to isolate the plasmid and then confirm the plasmid by sequencing with the primer.
For the T seven promoter, the plasmid should contain an N terminal translational fusion of the two FZ domains to MGFP. To begin protein purification. First transform BL 21 equalize cells with 100 nanograms of the purified plasmid.
Select for transformants on LB agar containing can mycin the next day inoculate A 10 milliliter culture of LB medium containing can mycin with a single colony from the overnight plate and incubate the culture overnight at 37 degrees Celsius with shaking, dilute the entire overnight colony into one liter of LB medium, supplemented with can mycin 0.2%glucose and 100 micromolar zinc chloride. Incubate the culture at 37 degrees Celsius with shaking until it reaches an optical density at 600 nanometers of 0.8 and then add IPTG to a final concentration of two millimolars to induce expression. After six hours of induction centrifuge the culture at 5, 000 times G for 10 minutes at four degrees Celsius to harvest the cells.
Re suspend the cell pellet in five milliliters of lysis buffer, and then lysis cells on ice with sunation using 50%power output and a five second on ten second off cycle for four minutes. Next, centrifuge cell lysate at 25, 000 times G for 30 minutes at four degrees Celsius, and then transfer the supernatant to a new collection tube on ice. To purify the protein, load the supernatant on a column that was pre-packed with one milliliter of nickel nitric acetic acid slurry.
After running the supernatant through the column, wash the resin with 20 milliliters of wash buffer. Elute the protein from the column with five milliliters of elution buffer, and then transfer the solution to dialysis tubing, dialyze the protein and storage buffer overnight after dialysis. Use a spin concentrator to concentrate the protein to at least 40 micromolar by centrifuging 3000 times G for one hour.
Determine the protein concentration and purity as indicated in the text protocol for storage. Aqua 200 microliters of the protein in 1.5 milliliter fuge tubes and cover the tube in foil to protect MGFP fusion proteins from photobleaching. Then flash, freeze the protein in liquid nitrogen and store at negative 80 degrees Celsius to prepare for protein transduction first preco the wells of a 24 well plate with 500 microliters of a solution of 50 micrograms per milliliter.
Polylysine for 30 to 60 minutes at 25 degrees Celsius. Aspirate the solution from the wells before seeding the cells culture. Heal a cells in DMM containing 10%FBS and 1%antibiotic anti mycotic at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide.
Harvest the cells and seed them onto the 24 well plate at an density of 200, 000 cells per well. Then place a 24 well plate in the incubator after about 24 hours of incubation when cells are 80 to 90%confluent. Aspirate the medium from each well and wash the cells with 500 microliters of prewarm serum free medium or SFM.
Add 250 microliters of SFM containing two micromolar Z-M-G-F-P protein and 100 micromolar zinc chloride to each well and then incubate the cells at 37 degrees Celsius for one hour. Next, aspirate the medium from the wells and wash the cells three times with 500 microliters of calcium and magnesium free docos phosphate buffered saline known as DPBS supplemented with 0.5 milligrams per milliliter of heparin. Next, add 200 microliters of 0.05%tripsin EDTA to the wells and incubate at 37 degrees Celsius for two minutes to detach the cells from the plate.
Transfer the detached cells to a new tube and centrifuge at 300 times G for five minutes. Then resuspend the cells in 500 milliliters of DPBS supplemented with 1%FBS measure the mean fluorescence intensity of the sample by flow cytometry using the fite channel as previously described. Adjust the forward and the side scatter to place the population of interest on the scale.
Next, use defined gating to analyze the control and the test samples. Collect 10, 000 live vent per sample and use the flow cytometry analysis software to process the data. Normalize the fluorescent intensity of protein treated cells to the control serum treated cells.
Zif MGFP fusion proteins were produced in e coli purified and analyzed by SDS page. These results show that the two fingers ZIF MG FP fusion protein was purified to greater than 95%homogeneity. When helo cells were treated with increasing concentrations of the ZIFF MG FP fusion protein.
They displayed a dose dependent increase in mean fluorescence intensity as measured by flow cytometry consecutive treatments with the Z MG FP fusion protein at a concentration of two micromolar resulted in nearly 100%of cells with MGFP fluorescence. After watching this video, you should have a good understanding of how to use the thief protein transaction domains to deliver functional proteins into my cells.
