The overall goal of this method is to isolate the G-quadruplex helicase G4 Resolvase1 protein from a bacterial expression system using a unique ATP dependent purification step to isolate nearly pure and catalytically active enzyme. This method can help researchers obtain nearly pure and catalytically active recombinant G4 Resolvase1 protein for us in a wide of in vitro biochemical assays. The main advantage of this technique is that it excludes misfolded or otherwise catalytically inactive enzyme from the protein preparation.
The idea to use ATP to elute the G-quadruplex helicase originated after being unable to elute the catalytically active enzyme off the G4 beads, even at exceptionally high salt concentrations. Prepare and grow large bacterial cultures expressing G-quadruplex Resolvase1 or G4R1 as described in the text protocol. Thaw the bacterial pellet in three milliliters of TN buffer at room temperature.
Hold the bottles in hand and swirl to thaw and resuspend the bacterial pellet. Once thawed and relatively evenly suspended bring the volume up to five milliliters with TN buffer. Next, dissolve 20 milligrams of lysozyme in 250 microliters of water per culture and add to the resuspended bacteria.
Allow the lysozyme to digest the bacteria for approximately five to 10 minutes while swirling the bottles in hand. The color of the suspension will begin to lighten slightly as the digestion proceeds, and the suspension will be relatively non viscous. Add 250 microliters of protease inhibitor cocktail abbreviated PIC.
Then, add one 10 microliter aliquot of leupeptin and mix thoroughly. Place the suspension on ice and add 10 milliliters of cold TN buffer and 22 microliters of beta mercaptoethanol or BME. Transfer the suspension to a 50 milliliter tube.
Sonicate the bacteria on ice with a digital sonicator set to 30%amplitude. Pulse at two seconds on and two seconds off for one minute. Repeat the sonication three times, allowing the samples to cool on ice for at least two minutes between sonication steps.
Next, add an equal volume of cold four X saline sodium citrate or SSC plus BME, PIC, and leupeptin and mix the suspension. In a tabletop centrifuge pre cooled to four degrees Celsius centrifuge the lysates at 2, 300 times G for 20 minutes. Then transfer the supernatant to fresh pre cooled 50 milliliter tubes.
Take one milliliter of streptavidin paramagnetic beads, or SPB suspension per culture, and transfer it to a 1.5 milliliter microcentrifuge tube. Pellet the SPB with a magnet. Then wash the SPB twice with two X SSC plus five millimolar EDTA PH8.
Next, resuspend the washed SPB in 200 microliters of the same solution. Add one three OD aliquot of G4 DNA to the SPB suspension. Mix quickly by pipetting up and down several times.
Then, place the tubes on a rotator and rotate at room temperature for at least 30 minutes. After this period of rotation, block the G4 bound SPB by adding one milliliter of 0.4%lactalbumin, and keep on ice until needed. To bind histidine tagged recombinant G4R1 to cobalt beads add one milliliter of cobalt bead slurry to each 50 milliliter tube containing the clarified bacterial lysate.
Incubate the mixture for 20 minutes at room temperature on a rotator. Following incubation spin down cobalt beads at 110 times G for five minutes in a four degrees Celsius tabletop centrifuge. Aspirate the liquid carefully, and leave a couple milliliters of liquid so as not to disturb the pelleted cobalt beads.
Then, wash the beads with 10 to 15 milliliters of cold four X SSC plus BME, and pellet the beads as before. Pour the next clarified lysate onto the pelleted cobalt beads. Incubate for a further 20 minutes at room temperature on the rotator, and wash the beads with 10 to 15 milliliters of four X SSC plus BME.
Then, pellet the beads at 100 times G for five minutes at four degrees Celsius. After a second wash, aspirate the liquid and leave about two milliliters of beads and liquid at the bottom of the tube. Transfer the protein bound cobalt beads to a pre cooled two milliliter tube by using a one milliliter wide bore pipette tip.
Briefly centrifuge the beads in the two milliliter tube at high speed in a microcentrifuge at four degrees Celsius. Gently remove and discard the supernatant by pipetting being careful not to lose the protein bound cobalt beads. Pipette 0.5 milliliters of histidine elution buffer, or HEB onto the beads and resuspend them by inverting the tube.
Then incubate the cobalt beads in HEB on a rotator for five minutes in a cold room. Next, centrifuge the suspension at 18, 000 times G in a four degrees Celsius microcentrifuge for one minute. Gently remove the protein containing supernatant by careful pipetting leaving a small amount of liquid on top of the cobalt beads.
Transfer the supernatant to a pre cooled 15 milliliter tube. Repeat these steps for a total of three HEB elutions. Then elute once with 0.2 molar EDTA at PH 6.0.
The color of the cobalt beads will change from pink to white as the EDTA chelates the cobalt. After this fourth and final elution has been transferred to the 15 milliliter tube, pipette the residual elution buffer from the cobalt beads by using a gel loading tip on the end of a one milliliter pipette tip. By plunging the tip to the bottom of the tube residual protein containing elution buffer can be collected.
Pilot the prepared G4 SPB to the side of the tube with a magnet and discard the supernatant. Then, add one milliliter of three X res buffer to the G4 SPB and pipette to mix. Pipette the G4 SPB suspended in three X res buffer into the 15 milliliter tube containing approximately two milliliters of protein containing HEB EDTA.
Incubate for 15 minutes in a 37 degree Celsius water bath with occasional agitation so the beads do not settle. After washing the protein bound G4 SPB as described in the text protocol pellet the G4 SPB with a magnet on ice and resuspend the beads in 100 microliters of pre warmed elution buffer. Immediately transfer the beads to a pre warmed PCR tube and incubate for 30 seconds at 37 degrees Celsius.
Promptly add 12 microliters of five molar sodium chloride and pipette up and down vigorously 20 to 30 times. The combination of the high salt and ATP contained in the elution buffer serves to elute recombinant G4R1 from the G4 beads. Immediately pellet the G4 SPB with a magnet, and transfer the protein containing elute to a fresh PCR tube on ice.
After repeating the elution and transfer the end product is approximately 200 microliters of elution buffer containing purified recombinant G4R1. Finally, perform the quality control enzymatic activity assay to test whether highly active recombinant G4R1 has been purified as described in the text protocol. The G4 assay provides a measure of recombinant G4R1 enzymatic activity.
Within the 0.2 to 0.013 microliter range in the enzyme it is typically observed that 50%of 0.2 picomoles of tetramethylrhodamine labeled tetramolecular G4 DNA is converted into monomers. Coomassie standing of purified recombinant G4R1 indicates a single band at the expected 120 kilodalton size with a minor contaminating band at approximately 75 kilodaltons. This band may represent truncated recombinant G4R1 that has maintained is ability to bind G4 DNA beads into elute in an ATP dependent manner.
The band of interest is quantified against a protein standard curve. This protocol typically obtains 200 microliters of 20 to 100 nanomolar purified enzyme per one liter of bacteria culture. After watching this video, you should have a good understanding of how to purify G4 Resolvase1 protein from a bacterial expression system using a unique ATP dependent purification step to isolate nearly pure and catalytically active enzyme.
Once mastered, and with prior preparation of the frozen bacteria pellets, the purification of recombinant G4R1 can be performed in approximately six hours. To maintain the optimal catalytic activity of the enzyme it's important to keep the preparation on ice, unless otherwise noted. Always use protease inhibitors.
Avoid bubble formation in the tubes. Freeze purified enzyme quickly, and avoid unnecessary freeze thaw cycles. After its development, this technique paved the way for researchers in the field to accurately define the binding affinities and helicase activities of this protein to a wide range of DNA and RNA substrates.
This method can also be adapted for the purification of other ATP dependent nucleic acid enzymes for which the product of the enzymatic reaction is no longer a binding substrate for the enzyme.
