Our protocol yields highly active PCD free from detectable nuclease, which can be used in any solution where aqueous oxygen is a contaminant. The techniques we use are easy to replicate and ensure that no nuclease contaminants are present in the product. To begin, combine one microliter of pVP91A-pcaHG PCD expression plasmid and 20 microliters of commercially available E.coli BL21 in a tube.
Flick the tube to mix, and place the tube on ice for five minutes. To continue the transformation reaction, place the tube in a water bath at 42 degrees Celsius for 30 seconds, then on ice for two minutes. Add 80 microliters of SOC media, and shake at 225 rpm and 37 degrees Celsius for one hour.
Next, pour the transformation reaction mixture onto an LB-ampicillin agar, and use a plate spreader to spread the mixture. Cover the plate with a lid, and incubate the plate at 37 degrees Celsius for 16 to 18 hours. After that, pick one colony from the plate, and inoculate in 50 microliters of LB-ampicillin in a 250-milliliter Erlenmeyer flask.
Place the flask on a shaker at 225 rpm to incubate at 37 degrees Celsius for 16 to 18 hours. Then, transfer 20 milliliters of the incubated culture to a four-liter flask filled with one liter of LB-ampicillin. Shake at 225 rpm at 37 degrees Celsius.
Every hour, draw one milliliter of the culture into a cuvette to measure the optical density at 600 nanometers on a photometer. As the culture optical density increases near 0.5, increase the frequency of measurements to every 15 minutes, until the OD600 reaches 0.5. Then, transfer the four-liter flask to a bin of ice.
Swirl the flask in the ice bath to reduce the culture temperature. Transfer the four-liter flask to an incubator at 17 degrees Celsius and 180 rpm. Continue to monitor the OD600 every 20 minutes.
At 0.7 OD600, add IPTG and ammonium iron sulfate hexahydrate stock solutions to reach a final concentration of 0.5 millimolar and 10 milligrams per liter, respectively. Shake the culture at 180 rpm and 17 degrees Celsius for 18 hours. After incubation, place the culture flask in ice for two minutes.
Harvest one milliliter of the induced cells in a 1.5-milliliter polypropylene microcentrifuge tube for SDS-PAGE analysis. Next, pour the bacterial culture into bottles, 500 milliliters each bottle, for centrifugation. Pellet the culture at four degrees Celsius and 3, 000 times g for 20 minutes.
Decant the supernatants. Pipette to resuspend the pellets, each in 12.5 milliliters of cold PBS, and combine every two resuspensions in a 50-milliliter conical tube. Pellet the cells again as previously.
Then, discard the supernatants, and use a pipette to add 10 milliliters of lysis buffer in each tube to resuspend the cells. After sonicating the tubes with induced cells, pour the bacterial lysate into a pre-chilled polycarbonate bottle. Centrifuge for 60 minutes at 120, 000 times g and four degrees Celsius, and pour the supernatant to a cold 50-milliliter conical tube.
After preparing FPLC instrument with the nickel-charged resin column, load the sample to the column at 0.15 milliliters per minute. Wash the column with 20 milliliters of nickel buffer at 20-millimolar imidazole. Retain the wash in a 50-milliliter tube for analysis.
Next, wash the column with 15 milliliters of nickel buffer at 125-millimolar imidazole. Collect the elution in 19 fractions of 0.8 milliliters each. Wash the column again with 15 milliliters of 100%nickel buffer B, and collect an additional 75 fractions of 0.2 milliliters each.
Continue with the analysis of the collected fractions on 12%SDS-PAGE gels to confirm presence of PCD. First, add 35 microliters of reaction buffer to a 1.5-milliliter polypropylene microcentrifuge tube. In the tube, combine five microliters of the peak chromatography fractions and 500 nanograms of three-kilobase pair supercoiled plasmid pXba.
Place the tube in a water bath at 37 degrees Celsius for one hour. After preparing agarose gel according to the manuscript, add 30 microliters of each reaction into wells in the gel. Electrophorese at 10 volts per centimeter at ambient temperature for approximately one hour.
Now, the orange G dye has reached the end of the gel. Immediately, use a fluorescence scanner to image the ethidium bromide signal of the gel. With the function of the intensity and image pixel size, determine the pixel volume of the various DNA species, such as supercoiled, linear, and nicked circles.
To determine the percentage of each DNA species, divide the pixel volume of a single DNA species by the total pixel volume for all individual species. The SDS-PAGE analysis shows the second elution peak contains minimal protein contamination, which represents nearly pure PCD heterodimer and minimal to undetectable nuclease activity. Thus, combine these 10 fractions from the second PCD elution in a five-milliliter conical tube for further analysis.
After size-exclusion chromatography purification of PCD, in a four-degree Celsius cold room, combine in a microcentrifuge tube appropriate volume of sodium chloride, Tris hydrochloride, magnesium chloride, dithiothreitol, PCA, and supercoiled plasmid pXba to make a 5X stock. Place a 96-well flat-bottom plate on ice, and transfer 10 microliters of the combined 5X solution to the wells. Immediately before analysis, add to each well 10 microliters of the individual PCD SEC fractions.
Set the plate reader to an internal temperature of 37 degrees Celsius, and transfer the 96-well plate to the plate holder. Retract the plate holder into the instrument, and measure absorbance at 290 nanometers at 20-second intervals for one hour. Set the instrument to shake the plate five seconds before each reading.
After one hour, add 10 microliters of stop solution to each well to terminate the reactions. Perform gel electrophoresis for the PCD SEC fractions. Select fractions with the most PCA oxidation activity, indicated by decreased absorbance and no observed nuclease contamination.
Measure the absorbance at 280 nanometers, and calculate the total PCD concentration based on the absorbance and the extinction coefficient. Store the selected fractions for long-term storage at minus 80 degrees Celsius. In this experiment, it was found that recombinant PCD, a heterodimer of hexahistidine tagged pcaH and pcaG, was expressed in E.coli.
The heterodimer was first purified by nickel affinity chromatography. The absorbance is shown in blue, and the percent concentration of nickel buffer B is shown in red. The flow-through shows the soluble bacterial proteins that did not bind to the nickel resin.
Some PCD eluted in the presence of 125-millimolar imidazole, but the majority of the protein eluted in 250-millimolar imidazole. Representative SDS-PAGE analysis indicates the fractions from the elution step were free of contaminants. Agarose gel of nuclease assay reveals contamination through comparing with the negative control without added protein and a positive control contaminated with a DNA nuclease.
Quantitation of the various DNA species observed in the agarose gel nuclease assay shows fractions 29 through 38 had little nuclease activity, which were chosen to be combined, concentrated, and further purified by SEC. Data from three representative SEC fractions showed that purified PCD reduced the absorbance at 290 nanometers, indicating oxidation of PCA. Following this procedure, the purified PCD can be used and optimized in the desired oxygen scavenging system.
This will determine the exact amount of PCD required for the system. This technique has been used to prolong fluorophore lifetimes in single molecule microscopy experiments, which has allowed for longer periods of data collection. The ultracentrifuge used is hazardous.
Be sure that centrifuges are properly balanced prior to spinning. Ethidium bromide is a hazardous reagent. Avoid contact by wearing personal protective equipment, and dispose of it appropriately.
