Protein Purification by InverseTransition Cycling

Protocol

1. Lyse the cells. Any lysis method should work, although ultrasonic disruption may help to break apart and resolubilize any ELP aggregates that have formed during culture. If sonication is used, lyse on ice to ensure that the solution temperature remains below the ELP Tt
(e.g., ~5°C or more lower than the Tt ).

2. Centrifuge the lysed cell suspension at ~20,000g for 15–20 minutes at ~4°C. Remove the supernatant to a new centrifuge tube and discard the pellet, which contains insoluble cellular debris.

3. Optional: Add polyethylenimine to a final concentration of 0.5% and mix gently. The solution should turn white due to coprecipitation of the polyethylenimine and the DNA present in the suspension. Incubate for 10–20 minutes on ice, occasionally mixing the suspension gently. Centrifuge at ~20,000g for 15–20 minutes at 4°C. Remove the supernatant to a new centrifuge tube and discard the pellet, which contains precipitated DNA and insoluble cellular debris.

I believe PEI can not be used for basic proteins.

Precipitation of nucleic acids with polyethylenimine prior to induction of the ELP inverse transition is recommended because some nucleic acids present in the soluble lysate can pellet with the ELP in the centrifugation steps of ITC, particularly when higher salt concentrations are used (e.g., greater than ~1.5 M NaCl). If the polyethylenimine precipitation step is omitted, separation from nucleic acids can still be
achieved using additional rounds of ITC.

Inverse Transition Cycling

4. Increase the solution temperature and/or add NaCl to trigger the inverse transition. The solution temperature and NaCl concentration required to trigger the transition depend on the ELP sequence and molecular weight, the fusion partner, and ELP concentration. The goal is to lower the ELP Tt at least 3–5°C below the solution temperature during centrifugation. An effective Tt of 25–30°C (achieved using NaCl concentrations in the 0.1–2 M range) and centrifugation at 35–40°C typically works well, although higher temperatures may be used if the target protein is thermally tolerant. However, fusion to the ELP can reduce the thermal denaturation temperature of the target protein, and, thus, care must be taken not to exceed this temperature during ITC. If irreversible precipitation of the fusion protein is observed during ITC, consider using a different combination of temperature and NaCl concentration to prevent denaturation of the target protein, which is a separate event from the ELP transition. When working with a new construct for which the precise Tt is unknown, it is helpful to increase the temperature and/or NaCl (e.g., in 5°C or 500 mM increments) slowly until the transition is observed in the cell lysate. If solid NaCl crystals are used, dissolve them gently by inverting the tube. Do not vortex vigorously, because the ELP fusion protein can aggregate at the air–water interface of bubbles and be subsequently lost when the supernatant is decanted.

The solution should become turbid because of aggregation of the ELP. Before proceeding to the centrifugation (step 5), it is essential that (1) the ELP fusion protein has undergone its transition and (2) the solution temperature is equal to or less than the centrifugation temperature. If both of these conditions are met, the ELP fusion protein will remain aggregated during the centrifugation and therefore will be captured. If, on the other hand, the centrifugation temperature is lower than the precentrifugation solution temperature, some or all of the ELP fusion protein may resolubilize and remain in the supernatant.

If no turbidity is observed after increasing the temperature and/or adding salt, confirm expression levels by SDS-PAGE. Although not generally required, spiking of additional free ELP (e.g., to a concentration of ~10–25 μM) with a matched Tt may be necessary for poorly expressed
fusion proteins. If the protein is highly overexpressed but turbidity is still not observed, it may be necessary to use an ELP tag either with a lower Tt or one with a greater molecular weight.

5. Centrifuge at a temperature above the Tt at ~15,000g for 5–10 minutes to pellet the aggregated ELP fusion protein.

Typically this centrifugation is completed at a temperature in the range of ~25–40°C.

6. Discard the supernatant, which should be clear, and resuspend the pellet in a buffer of choice that is at a temperature below Tt.
The pellet is often brown or off-white during the first round of purification from cell lysate but is typically translucent in later rounds of ITC. The pellet can be resuspended by working aggressively with a pipettor (a 1000-μl pipettor with disposable tips is recommended). Place the pipette tip near the pellet, and repeatedly withdraw and expel the solution over the pellet. The aggregated ELP fusion protein is also typically adherent to the centrifuge tube, and therefore the tube walls should be washed thoroughly. A period of soaking (again, at a temperature below Tt ) can help in the resolubilization. Mechanical scraping of the pellet and tube walls may also be helpful. Ensure that the pellet has been entirely resuspended. During the first round of ITC from the cell lysate, the solution typically remains turbid after resuspension of the pellet due to insoluble contaminants that will be removed in step 7. However, for later rounds of the ITC, the solution is generally clear after dissolution of the pellet.

The pellet can be resuspended in any desired volume (~1/50 to 1/10 of the lysate volume is recommended, depending on the expression level of the target protein).

7. Centrifuge at ~20,000g for 10–15 minutes at 4°C to remove any remaining insoluble matter.

Retain the supernatant, and discard any pellet that is formed.

For the first round of purification from cell lysate, the pellet from this cold centrifugation step can be relatively large. However, it typically does not contain significant amounts of the ELP fusion protein.

8. Repeat steps 4–7 at least one additional time.

Typically, two rounds of transition cycling provide a high level of purity, although additional rounds may be undertaken if desired. Also note that a lower salt concentration can generally be used for the second round because the transition temperature is lowered by the significantly
increased ELP fusion protein concentration achieved in the first round.

9. Optional: If a protease recognition site has been incorporated in the primary amino acid sequence between the ELP and the target protein, free target protein can be obtained by incubating the purified ELP fusion protein with the protease to cleave the ELP tag.

The solubility of the ELP fusion protein can be significantly greater than the solubility of the free target protein. Therefore, ensure that the protease cleavage step is performed at a concentration within the solubility limits of the free target protein. If the protein concentration is too
high, addition of the protease and subsequent cleavage of the ELP can cause irreversible precipitation of the liberated target protein. Also note that, unless a secondary purification is performed, the protease remains with the purified target protein. Although not currently commercially available, the use of ELP-tagged proteases could solve this problem by enabling the simultaneous removal of both the protease and the cleaved ELP tag from solution by ITC, leaving only the purified target protein in the retained supernatant.

A final round of ITC can then be used to remove the free ELP, retaining the purified, free target protein in the supernatant.

10. Purified ELP fusion proteins can often be stored frozen in water at –80°C until use. Use a small aliquot to test for denaturation of the target protein after freezing; if the protein denatures, storage at –20°C in a 50% glycerol solution may be necessary.

Buffers

NaCl: Expression culture harvested and resuspended in low-salt buffer (e.g., Tris or phosphate buffered at pH 7–7.5 with 20–150 mM NaCl) in ~1/20 of the culture volume.

PEI25K Polyethylenimine, 10% aqueous solution