Purification of Prototype Foamy Virus Intasomes by Size Exclusion Chromatography

Begin with a suspension containing precipitates of prototype foamy virus or PFV intasomes, monomeric integrases, DNA oligomers, and protein aggregates.

PFV intasomes are tetramers of viral integrases complexed with synthetic oligomers that mimic viral genomic DNA.

Add a salt solution to the suspension and incubate on ice. The salt reduces the electrostatic interaction between the intasomes, enabling the precipitates to solubilize.

Pipette gently at regular intervals to promote intasome solubilization.

Centrifuge to pellet remaining precipitate and collect the supernatant containing soluble intasomes.

Take a pre-equilibrated size-exclusion chromatography column packed with cross-linked agarose beads and load the supernatant onto the column.

Due to their large size, protein aggregates and intasomes bypass the bead pores and elute earlier.

Smaller monomeric integrases and oligomers enter the bead pores and elute later.

Collect the early-eluting fractions containing intasomes for further downstream analysis.

Prepare two wide-bore pipette tips by removing two to four milliliters from the end of a 200 microliter tip with a new razor or scalpel blade on a clean surface. Remove the dialysis tubing from the dialysis buffer. To prevent dilution of the intasome sample with dialysis buffer that may remain at the end of the tubing near the clip, use a micropipette with a small pipette tip to remove any excess dialysis buffer.

Remove the clip from this end of the dialysis tubing. When recovering the assembly, it is important to collect as much of the solution and precipitate as possible. Use the wide-bore pipette tip to transfer the sample, including the precipitate, inside the dialysis tubing, to a 1.5 milliliter tube on ice.

Note the total volume of the recovered material. The total volume is typically approximately 140 microliters. The sample with precipitate is at 200 millimolar sodium chloride.

Increase the salt to a final concentration of 320 millimolar sodium chloride by adding the appropriate volume of a stock five molar sodium chloride solution. Transfer the ice bucket with the sample into a four degree Celsius cold room. Use the wide-bore pipette tip to resuspend and solubilize the precipitate by pipetting.

Repeat every 20 minutes for at least one hour. Most of the precipitate should go into solution. To purify the intasome, equilibrate a cross-linked, agarose size-exclusion chromatography or SEC column with SEC running buffer at a flow rate of 0.4 milliliters per minute.

Centrifuge the intasome sample in a microfuge at 14,000 times g for 10 minutes at four degrees Celsius to pellet any remaining precipitate. After carefully removing the supernatant load it onto a 200 microliter injection loop. Apply the sample to the SEC column.

Elute with 25 milliliters of SEC running buffer and collect 95 fractions of 270 microliters. Observe that the SEC chromatogram displays three A280 and A260 peaks.