The overall goal of this procedure is to purify recombinant elastin like polypeptides from e coli by inverse transition cycling. This is accomplished by first collecting the lysate from e coli cells that have expressed recombinant elastin like polypeptide, and removing insoluble cellular debris by centrifugation. The second step is to remove soluble contaminants by inducing the elastin like polypeptide transition with the addition of salt or heat.
Then collecting the elastin like polypeptide coate by centrifugation and discarding the supernatant. This step is termed a hot spin. Next, the resuspended elastin like polypeptide solution is centrifuge to remove the insoluble contaminants.
The soluble elastin like polypeptide in the supernatant is retained and the pellet is discarded. This step is termed a cold spin, repeat cycles of hot and cold spins until satisfactory purity of the elastin like polypeptide is achieved. Ultimately, inverse transition cycling yields pure elastin like polypeptide products that can be characterized for use in a variety of biomedical applications.
Elastin like polypeptides purified by this technique have a number of useful applications, including biosensing, tissue engineering and drug delivery, where the transition temperature and biopolymer architecture of the elastin like polypeptide can be specifically tuned for each application. To begin, express the desired ELP encoding plasmid in e coli using the conditions described in the accompanying text protocol. After shaking a one liter culture in an incubator for 24 hours at 37 degrees Celsius, transfer the culture to a one liter centrifuge bottle, and then centrifuge the bottle at 2000 Gs for 15 minutes at four degrees Celsius.
Next, discard the supernatant and resus. Suspend the pellet in PBS water or other desired buffer to a total volume of 45 milliliters. Then transferred the cell suspension to a 50 milliliter conical tube.
Place the cell suspension on ice and once cooled sonicate for a total of nine minutes in cycles of 10 seconds on and 20 seconds off. With an output power of 85 watts. Allow the sample to cool on ice for approximately 10 minutes following the first round of sonication, and then repeat the nine minutes sonication cycle.
Transfer the lysate to a 50 milliliter round bottom centrifuge tube. Then add two milliliters of 10%weight per volume polyethylene for each liter of culture, and shake the tube to mix next, centrifuge the tube at 16, 000 GS for 10 minutes at four degrees Celsius. Then transfer the supernatant to a clean 50 milliliter round bottom centrifuge tube and discard the pellet.
Add crystalline sodium chloride to the sample to induce the ELP transition. Once the salt has dissolved, the solution will turn cloudy, which indicates the phase separation of the ELP from the solution. Then pellet the ELP by centrifuging the sample at 16, 000 GS for 10 minutes.
At room temperature, the ELP pellet may at first appear opaque, discard the supernatant, and then add one to five milliliters of the desired buffer after cooling. The ELP pellet may appear translucent and brown in color. Resus suspend the pellet by pipetting or rotating the tube at four degrees Celsius.
Next pipette one milliliter aliquots of the resuspended ELP solution into clean 1.5 milliliter micro centrifuge tubes. Centrifuge the tubes at 16, 000 GS for 10 minutes at four degrees Celsius. Then transfer the supernatant to a clean tube and discard the pellets.
Induce the ELP transition with the addition of sodium chloride. If the ELP has a high transition temperature that cannot be induced with salt alone, incubate the sample on a heat block for 15 minutes at a temperature above the ELP transition temperature. Then centrifuge the sample at 16, 000 GS for 10 minutes at a temperature above the temperature used for the ELP transition step.
Discard the supernatant and resuspend the ELP pellet in 500 to 750 microliters of desired buffer by pipetting or rotating the tube at four degrees Celsius until the pellet is re solubilized. Next, centrifuge the sample at 16, 000 GS for 10 minutes at four degrees Celsius to pellet any insoluble contaminants. Then transfer the snat to a clean tube and discard the pellet Repeat inverse transition cycling until no contaminant pellet is observed.
This typically requires two to five cycles. Prepare a dilution series of ELP solutions in PBS or other desired buffer. Then use a temperature controlled UV v spectrophotometer to measure the optical density at 350 nanometers over a range of temperatures while heating the sample at a rate of one degree Celsius per minute.
If no increase in optical density is seen, then the transition temperature may exceed the upper temperature limit. Next, measure the decrease in optical density over the same range of temperatures at a cooling rate of one degree Celsius per minute. To verify the reversibility of the ELP transition plot, the optical density versus the temperature, then determine the transition temperature of the homo polymer ELP.
By identifying the inflection point on this turbidity profile, the successful purification of LPs is confirmed by SDS page as can be seen here. The number of contaminants is much greater in the initial e coli lysate as compared to the sample after one round of inverse transition cycling. The ELP transition temperature is characterized by temperature programmed turbidity for an ELP homo polymer.
The turbidity profile exhibits a single sharp increase in the optical density, which defines the transition temperature at a given concentration. The reversible nature of the ELP transition is confirmed with a cooling turbidity scan. ELP die block Copolymers exhibit a more complex turbidity profile than homo polymer LPs.
Dynamic light scattering measurements can then be taken to confirm the behavior of these LPs that is inferred from the turbidity profile file. After watching this video, you should have a good understanding of how to purify recombinant elastin like polypeptides from e coli using inverse transition cycling. No, no, No, no, no.
