JoVE Encyclopedia of Experiments
Microbiology
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Begin with a suspension of bacterial cells expressing polyhistidine-tagged viral integrase.
Sonicate the suspension to disrupt the bacterial membranes and release the integrase.
Transfer the lysate to an ultracentrifuge tube.
Centrifuge at high speed to pellet the cell debris and collect the supernatant.
Next, take a chromatography column packed with nickel-charged resin and equilibrate it with buffer.
Load the clarified lysate onto the column.
Proteins with histidines bind weakly to nickel, while polyhistidine-tagged integrase binds strongly.
Wash with buffer to remove unbound proteins, then add elution buffer with a linearly increasing imidazole concentration.
Imidazole competes with histidine for nickel binding and releases the integrase.
Collect the fractions and transfer aliquots to tubes containing loading buffer.
Heat to denature the proteins, then load onto a polyacrylamide gel.
Perform electrophoresis and analyze the protein bands.
Identify fractions containing pure integrase, without any contaminating proteins, and pool them to obtain purified viral integrase.
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 real time 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 eluting 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.