The overall goal of this procedure is to produce large amounts of highly active HSP 1 0 4 A protein deaggregate. This is accomplished by first expressing HSP 1 0 4 in code on optimized e coli cells. The HS P 1 0 4 protein is then purified through the use of a histidine affinity tag, followed by an ion exchange column.
After ion exchange chromatography. The histidine tag is cleaved using TEV protease. The final purification step is to run the cleaved HSP 1 0 4 protein through a gel filtration column.
Also, ultimately, Elif reactivation assay is used to determine whether active HSP 1 0 4 has been purified. The main advantage of this technique over existing protocols is both a small number of steps in the large yield of highly active HSP 1 0 4 that is suitable for structural and mechanistic studies. Individuals new to this method may struggle because the majority of purification steps occur in a single day, which results in a demanding schedule.
Before beginning this protocol prepare all necessary buffers for HSP 1 0 4 purification as described in the written protocol accompanying this video. Also express the HSP 1 0 4 protein overnight in e coli following the procedure detailed in the text following expression, harvest the cells by centrifugation. Re suspend the cell pellets in 10 milliliters of pre chilled lysis buffer prior to cell lysis using a French press homogenizer ensure that all cell clumps have been solubilized After equilibrating, the homogenizer with lysis buffer lyce the cells with two passes through the cylinder.
At a pressure of 15, 000 to 18, 000 PS, I remove the cell debris by centrifugation and retain the supernatant for continued purification. Begin the purification of HSP 1 0 4 by mixing the supernatant with a 50%slurry of lysis buffer equilibrated nickel SRO speeds. Rotate the sample slowly for three hours at four degrees Celsius, such that the beads remain evenly suspended and frothing is minimized.
Following incubation, collect the nickel spheros by centrifugation. Wash the retrieved nickel spheros with 25 column volumes of wash buffer. Collect the beads again by centrifugation, after which the wash buffer can be removed by aspiration.
Then wash the beads with five column volumes of wash buffer with one molar potassium chloride, followed by 25 column volumes of wash buffer. To elute HSP 1 0 4. Mix the nickel sphero speeds with elution buffer and rotate at four degrees Celsius for 20 minutes.
Separate the beads from the eluate by centrifugation through empty 1.2 milliliter. Spin columns. Every liter of cells yields about 15 milligrams of HSB 1 0 4.
At this point in the purification, concentrate the eluate to approximately 1.5 milliliters using molecular weight cutoff 30, 015 milliliters. Centrifugal concentrator units. Then add 14.5 milliliters of buffer Q to dilute the protein tenfold.
Repeat this process three times to buffer. Exchange the protein before HSP 1 0 4 purification via anion exchange chromatography. Filter the protein through a low protein binding 0.22 micromolar syringe filter.
Then inject the protein onto a buffer Q equilibrated resource Q six milliliter column. Wash away weekly binding proteins with one column volume of 20%buffer B elute the HSB 1 0 4 with a linear gradient over five column volumes collecting one milliliter fractions, HSB 1 0 4 and most variants typically elute at about 400 millimolar sodium chloride. To remove the hexa histidine tag, exchange the protein using a centrifugal concentrator, cleve the tag with the TEV protease according to the manufacturer's instructions.
Performing cleavage at 30 degrees Celsius for two to four hours, followed by a 16 hour incubation at four degrees Celsius. Deplete any remaining hexa histidine tagged HSB 1 0 4 and TEV protease with nickel SROs by adding the beads in excess to the amount of HSP 1 0 4 in the cleavage reaction. Then remove the beads from the sample via centrifugation through empty spin columns.
To prepare for the final purification step, exchange the protein into size exclusion buffer using molecular weight cutoff. 30, 000 centrifugal filter units following filtration through a low protein binding 0.22 micromolar syringe filter. Further purify HSP 1 0 4 via size exclusion chromatography using a size exclusion buffer.
EQUILIBRATED super oh six column. Perform the FPLC run according to the manufacturer's instructions. After purification, pull the HSP 1 0 4 fractions and concentrate them in centrifugal concentrator units due to loss of material in separating HSP 1 0 4 degradation products and contaminants, the final yield of purified full length HS P 1 0 4 is approximately one to three milligrams per liter of starting culture.
Shown here is the SDS page analysis of the nickel seros affinity purification. The histidine tagged 105 kilodalton HSP 1 0 4 protein is visible in samples taken from the lysate, the bead load, the bead flow through, and the bead eit. Note that not all the HSP 1 0 4 is able to bind to the nickel spheros nickel affinity chromatography is followed by ion exchange on a resource queue column.
The blue trace represents absorbence at 280 nanometers and the green trace represents percent buffer B.The first peak, which eludes at 20%B, contains impurities degradation products and improperly folded HSP 1 0 4. The and major peak contains properly folded and active HSP 1 0 4 as indicated by SDS page analysis after ion exchange. The HEXA histamine HSP 1 0 4 protein is subject to cleavage of the hiss tag by pro TV protease cleavage.
Note that prot v cleaved HSP 1 0 4 migrates more rapidly than the hexa histidine and tagged protein. Cleaved HSP 1 0 4 was then further purified via AUP O six gel filtration column. The peak between the dashed lines represents the pooled fractions.
Shown here are results from the Luciferase reactivation assay, which is used to assess HSP 1 0 4 disaggregation activity denatured firefly luciferase aggregates were incubated with either HSP 40 and HSP 72, HSB 1 0 4 or HSP 1 0 4, HSP 40 and HSB 72. Denatured Luciferase is only fully reactivated in the presence of both HSP 1 0 4 as well as HSP 40 and HSP 72 as evidenced by the large increase in luminescence Once mastered. This protocol can be accomplished in two days if it is performed properly.
