The overall goal of this procedure is to demonstrate the steps involved in the construction and production of artificial ECM proteins or A ECM containing elastin like repeats. This is accomplished by first constructing recombinant genes that encode the A ECM proteins. Next, the recombinant genes are inserted into bacterial hosts.
At this point, the recombinant proteins are expressed in the bacterial hosts and then harvested following protein extraction through cell lysis. The final step is to purify the proteins using inverse transition cycling or ITC, which exploits the unique phase transition behavior of elastin. Like polypeptides.
Centrifugation cycles are done at four degrees Celsius and 37 degrees Celsius. Ultimately, gel electrophoresis and western blot techniques are used to identify the recombinant proteins and determine their purities. The main advantage of this technique over existing methods like affinity columns, is that we can extract large amount of proteins relatively cheaply.
Begin this procedure with all ligament design as described in the text protocol. Once the oligomers have been obtained, anil the DNA oligomers to obtain the desired gene sequence. First dissolve DNA oligonucleotides in DNA oligo buffer to a final concentration of one microgram per microliter.
Add four microliters of each all ligament to 32 microliters of DNA and kneeling buffer to achieve a total 40 microliter mixture. Then boil a beaker of water using a hot plate and immerse the mixture for five minutes. At 95 degrees Celsius, remove the beaker and gradually cool the entire setup in a styrofoam box overnight, the oligomers have now been in kneeled and are ready for digestion.
Add the corresponding restriction enzymes to digest the al oligomers and host vectors separately as described in the text Protocol Digest for three to four hours at 37 degrees Celsius. After running the digestion products on an agarose gel and purifying the DNA, combine the gene of interest sequentially by ligating the digested DNA insert into the plasmid vector with T four ligase. Using the recipe found in the text protocol, incubate the ligation mixture at room temperature for two hours.
Next, transform the cells using heat shock by Ali. Quoting 50 microliters of competent cells into clean pre chilled micro centrifuge tubes. Pipette five microliters of the ligation mixture into the cells pipetting gently up and down to mix.
Leave the mixture on ice for 20 minutes. Then immerse the micro centrifuge tube containing the cell mixture in a 42 degree Celsius water bath for two minutes and return to ice for two minutes. Add 500 microliters of SOC media into the micro centrifuge tube and incubate at 37 degrees Celsius with shaking for one hour.
Following incubation, spread 50 to 500 microliters of the cell ligation mixture onto a two times YT auger plate containing ampicillin that has been prewarm to room temperature. Incubate the plates upside down at 37 degrees Celsius overnight. The next day, pick DNA colonies from the auger plate using clean pipette tips.
Grow the colonies in five milliliters of two times. YT media containing ampicillin overnight at 37 degrees Celsius overnight with shaking the following day. Use a plasmid isolation kit according to the manufacturer's protocol to extract the DNA plasmids for each colony.
Picked elute, the DNA with 50 microliters of water after transformation of the recombinant plasmid into the bacterial expression host as described in the text protocol. Pick a colony from the transformed auger plate with a pipette tip inoculate. 10 milliliters of sterile, terrific broth or TB media containing both ampicillin and chloramphenicol antibiotics in a test tube.
Incubate this starter culture at 37 degrees Celsius overnight with shaking at 225 RPM. Then transfer 10 milliliters of the starter culture into one liter of fresh and sterile TB media. Supplemented with the same antibiotics in a three liter erlin Meyer flask.
Incubate the culture at 37 degrees Celsius with shaking at 225 RRP M for two to three hours. Observe the optical density of the culture at 600 nanometers or OD 600 to confirm that it has reached 0.6 to 0.8 by pipetting one milliliter of culture into an empty Q vet for reading. Next, induce the culture with IPTG to a final concentration of one millimolar and incubate at 37 degrees Celsius with shaking at 225 RPM for another four hours.
Harvest the cells by transferring the culture to one liter centrifuge, bottles and centrifuging at 12, 000 Gs for 30 minutes at four degrees Celsius. After discarding the supernatant weigh the cell pellets and resuspend in 10 buffer at 0.5 grams per milliliter. Freeze the resuspended cell culture at minus 80 degrees Celsius overnight.
The next day thaw the frozen cell culture in a water bath at room temperature or on ice. To L the cells add deoxy ribonuclease one ribonuclease a and phenyl methyl suen fluoride. While thawing the solution, homogenize the solution with slow stirring.
After all resuspended cells are thawed, adjust the solution to pH 9.0 to increase the protein solubility in water by very carefully. Adding six N sodium hydroxide dropwise with stirring on ice to achieve a homogeneous consistency. Then lice the cells by ultrasonic disruption for 20 minutes on ice, using a two millimeter diameter flat tip and a five second pulse.
Centrifuge the cell solution at 12, 000 Gs for 30 minutes at four degrees Celsius. Transfer the supernat to a clean, empty bottle and store at four degrees Celsius for purification later. Meanwhile, resuspend the cell pellet again with 10 buffer and save 20 microliters of the cell lysate.
For SDS page characterization, refreeze the resuspended cell pellet at minus 80 degrees Celsius to complete cell lysis. Repeat the freeze thaw and sonication process up to three times. Collate the cell lysate from earlier and proceed to purify the A ECM proteins using ITC in which centrifugation cycles are performed at different temperatures in high salt conditions.
Split the cell lysate into 50 milliliter centrifuge, bottles, and centrifuge at 40, 000 Gs for two hours at four degrees Celsius. Remove the supernatant by pipetting to get a clean separation from the pellet. Collect the supernatant in clean centrifuge bottles and add sodium chloride to a final concentration of one molar.
Warm the solution to 37 degrees Celsius for two hours with shaking at 225 RPM. Next centrifuge. The supernatant at 40, 000 Gs for two hours at 37 degrees Celsius.
After decanting, the supernatant crush the pellet using a metal spatula and resuspend the pellet bits in ice. Cold autoclave distilled water with vigorous stirring overnight at four degrees Celsius using a magnetic stir bar and plate until the pellet is completely dissolved. Repeat these cycles until the pellet at 37 degrees Celsius is white, at which point it can be dissolved in water overnight and centrifuge.
To obtain the protein solution, desalt the protein solution by dialyzing it against distilled water at four degrees Celsius, changing the water every four hours or eight hours for overnight for two to three days after saving 20 microliters of the purified protein for SDS page Lyophilize the rest of the purified protein as shown here. An intense protein band is at the predicted molecular weight in the induced sample. Also shown is the SDS page gel containing a purified protein sample with a molecular weight of 22 kilodaltons corresponding to the laminin five A ECM.
The estimated purity of the A ECM proteins is about 90 to 95%This value was derived by comparing ratios of the band intensities of the target protein and other protein bands present on the SDS gel. Finally, to determine if the purified protein was indeed the A ECM proteins, Western blotting was performed here. The protein purity of the three A ECM proteins is shown with an SDS page gel and compared against an image of the nitrocellulose membrane for western blotting.
The analysis reveals the presence of the target proteins collagen four A ECM, and laminate five A ECM tagged using anti hexa histidine tag antibodies conjugated with horse radish peroxidase. The final yields of the A ECM proteins ranged between 60 milligrams per liter to 120 milligrams per liter. After watching this video, you should have a good understanding of how to contract and purify proteins containing elastin light domains for your specific application.
