The overall goal of this protocol is to identify and remove contaminating nuclease activity during protein purification. This method can help answer key questions in the retrovirology field, such as biochemistry, structure, and dynamics of integration. The main advantage of this technique is that it allows identification and removal of contaminating DNA nuclease activity from a recombinant protein of interest during purification.
Begin by thawing one pellet of E.Coli expressing prototype foamy virus integrase on ice. Use a sonicator equipped with a 0.5-inch diameter biohorn with a 0.125-inch diameter tapered microtip to sonicate for 30 seconds at 30%amplitude with the tube on ice. After sonication, transfer the cell sample to a cold ultracentrifuge tube, and spin at 120, 000 times g for 60 minutes at four degrees Celsius.
Following centrifugation, there should be an obvious pellet, and the supernatant may have a yellow color. Transfer the supernatant into a cold 50-milliliter conical tube. Next, set up the 110-millimeter long, five-millimeter diameter FPLC column with a 1.5 milliliter nickel-charged resin.
First, connect the column to the FPLC instrument, and apply buffer. The instrument should be collecting realtime data of conductivity and UV absorbance at 280 nanometers. The conductivity and UV readings should stabilize.
If necessary, continue to flow buffers through the column until readings are stable. Using a superloop, load the protein sample onto the column at a flow rate of 0.15 milliliters per minute with a maximum pressure limit of 0.5 megapascal. Attach the superloop to the AKTA FPLC.
After washing the column and eluding the proteins, pick 15-microliter aliquots of the initial flowthrough, wash, and peak 280-nanometer absorbance fractions, and add 15 microliters of 2X SDS-PAGE sample buffer to each sample. Boil the samples for three minutes. Load 10 microliters of each sample onto 10%SDS-PAGE gels, along with four microliters of pre-stained protein standards.
Visually inspect the gel to identify fractions that appear to contain nearly pure prototype foamy virus integrase. Combine these fractions, and make a note of the volume while pipetting. This is typically eight to 10 milliliters.
Using a spectrophotometer, measure the UV absorbance at 280 nanometers of the combined fractions, and calculate the total amount of prototype foamy virus integrase protein. A typical yield is around 10 milligrams per liter of induced culture. To remove the hexahistidine tag, supplement the prototype foamy virus integrase with a final concentration of 10-millimolar DTT and 0.1-millimolar EDTA.
Then add 15 micrograms of human rhinovirus 3C protease per milligram of prototype foamy virus integrase, and incubate at four degrees Celsius overnight. To perform heparin affinity chromatography fractionation of the prototype foamy virus integrase, reduce the sodium chloride concentration of the sample to 200 millimolar by adding 1.5 volumes of buffer C.Then load the diluted prototype foamy virus integrase sample to the heparin Sepharose column at 0.5 milliliters per minute flow rate with a maximum pressure limit of one megapascal. After washing the column and collecting the gradient and final wash, analyze load, flowthrough, wash, and fractions by 8%SDS-PAGE.
Then evaluate fractions by staining the gel with Coomassie blue. Prototype foamy virus integrase and the contaminating nuclease have different affinities for heparin Sepharose chromatography. Identify heparin Sepharose fractions that appear to contain nearly pure prototype foamy virus integrase to use in the nuclease assay.
To begin the nuclease assay, combine three microliters of heparin fraction and 50 nanograms of three-kilobase supercoiled plasmid DNA and reaction buffer to a final volume of 15 microliters. Incubate at 37 degrees Celsius for 90 minutes. Stop the reaction with one milligram per milliliter Proteinase K and 0.5%SDS, and incubate at 37 degrees Celsius for one hour.
Then add 3.5 microliters of 6X loading dye to the nuclease assay reactions, and load the entire volume on a 1%agarose gel. Run the gel at 100 volts at ambient temperature for one hour or until the dye front reaches the end of the gel. When electrophoresis is complete, a fluorescent scanner image of the gel should show that linear DNA runs true to size at three kb, supercoiled DNA runs faster to around two kb, and that relaxed circle DNA runs slower at close to 3.5 kb.
Using image analysis software, calculate the pixel volume of the supercoiled, linear, and relaxed circle plasmid in each lane. Call the sum of the pixel volumes for these three DNA forms the total DNA value, and use it to calculate the percentage of linear and relaxed circles. Fractions that contain contaminating nuclease display a higher percentage of linear and relaxed circles compared to the negative control.
Combine the heparin Sepharose fractions that do not display contaminating nuclease activity, and transfer to 10 millimeter, six to eight kilodalton molecular weight cut-off dialysis tubing. Dialyze at four degrees Celsius overnight against one liter of dialysis solution. The next day, recover the nuclease-free prototype foamy virus integrase sample from the dialysis tubing.
Measure the absorbance at 280 nanometers, and calculate the final protein concentration. Aliquot the protein, and snap freeze in liquid nitrogen. Then store the aliquots at minus 80 degrees Celsius.
The nuclease assay was performed as outlined in this video. Negative controls include plasmid DNA with no protein and a previous purification of wild-type prototype foamy virus integrase known to be free of nuclease. A positive control for contaminating nuclease activity is a previous purification of catalytically inactive prototype foamy virus integrase that is known to have contaminating nuclease.
Following this procedure, other methods, like biochemical assays or single molecule imaging, can be performed in order to answer additional questions, like enzyme kinetics and dynamics. After watching this video, you should have a good understanding of how to identify and remove contaminating nuclease activity from a recombinant protein.
