This method can help answer key questions in epigenetics and TET2 mediated 5-Methylcytosine demethylation field such an analysis of activity of normal and clinical TET2 mutations. The main advantage of this technique is that native TET2 can be purified in a single step, and its activity can be analyzed in terms of formation of all three products. Several graduate students will be demonstrating the procedure.
The protein purification will be performed by Chayan Bhattacharya, the TET2 assay by Aninda Sundary Dey, and the LC-MS/MS analysis by Navid Ayon. For bacterial transformation, add one microliter of recombinant pDEST 14 destination expression vector containing untagged human TET2 dioxygenase to 100 microliters of chemically competent E.coli BL21 DE3 cells in a 1.7 milliliter tube. After incubation on ice for at least 15 minutes, heat shock the mixture at 42 degrees Celsius for 30 seconds in a water bath.
Immediately after heat shock, place the cells back on ice for a minimum of two minutes. Following this, add 250 microliters of super optimal broth with catabolite repression to cells. Incubate the bacterial cells for one hour at 37 degrees Celsius in a shaker.
After incubation, spin down the cells by centrifuging the tube at 9, 000 times gravity for one minute. Discard 70%of the supernatant by pipetting, and dissolve the pellet in the remaining media. Spread the cell suspension on a Lirua broth agar plate containing 100 micrograms per milliliter ampicillin.
Incubate the plate for 16 hours at 37 degrees Celsius. Select one isolated colony, and inoculate it into 10 milliliters of LB ampicillin media. Incubate the tube at 37 degrees Celsius in a shaker overnight.
Following this, use 100 microliters of bacterial culture to inoculate 100 milliliters of LB ampicillin media as a primary culture. Incubate the tube at 37 degrees Celsius in a shaker overnight. The next day, inoculate 15 flasks, each containing 600 milliliters of LB ampicillin media with six milliliters of primary culture per flask.
Incubate the flasks at 37 degrees Celsius on a shaker at 180 RPM. To check the density of the bacterial culture, measure its optical density at 600 nanometers, or OD600, using a spectrophotometer. After the culture reaches a density of 0.8 at OD600, induce the expression of TET2 protein with 300 microliters of one molar IPTG in each flask, and grow the culture for an additional 16 hours at 17 degrees Celsius.
Following incubation, transfer the bacterial culture to centrifuge bottles. Centrifuge the bacterial culture expressing the TET2 enzyme at 5, 250 times gravity for 45 minutes. Use the bacterial pellet for TET2 purification.
Working on ice or at four degrees Celsius, re-suspend the bacterial pellet in 100 milliliters of 50-millimolar MES buffer, pH six. Then take five milliliters of the cell suspension, and bring it to 45 milliliters with the buffer. Sonicate the suspension for five times 30 seconds at power 20, with 60-second cooling intervals.
Spin the lysate at 5, 250 times gravity for 45 minutes. Collect the supernatant containing the soluble TET2 enzyme, and pass it through 0.45 micron filters before loading onto an FPLC system. Pack 30 milliliter of a strong cation exchange resin into an FPLC column.
Equilibrate the column with 10 bed volumes of wash buffer at the constant flow rate of 0.3 milliliters per minute using an FPLC system. Load the clarified lysate onto the pre-equilibrated column, and wash with approximately 10 bed volumes of wash buffer until the flow-through becomes clear. Elute TET2 using a zero to 10%gradient from the wash buffer to the elution buffer in 15 bed volumes, followed by a hold at 10%elution buffer for two bed volumes.
Collect 100 microliter samples of cell lysate before and after column loading, along with all the elution fractions, and analyze on 10%resolving SDS-PAGE gel. Pool the fractions containing TET2 protein, and freeze-dry the protein. Then dissolve the enzyme in 10 milliliters of water, and store at minus 80 degrees Celsius.
Perform all demethylation reactions in triplicate with three micrograms of substrate. Add 100 micrograms of purified TET2 enzyme to 50 microliters of total reaction buffer. Following one hour incubation at 37 degrees, quench the TET2 catalyzed oxidation reactions with five microliters of 500-millimolar EDTA.
To prepare samples for analysis, separate the DNA from the TET2 reaction mixture by first adding 100 microliters of Oligo binding buffer to 55 microliters of quenched reaction. Following this, add 400 microliters of 100%ethanol to the mixture. Pass this mixture through an Oligo binding column.
After washing the bound DNA with 750 microliters of wash buffer, elute the DNA in 20 microliters of water. Now, digest the isolated DNA with two units of DNase I and 60 units of S1 nuclease at 37 degrees Celsius for 12 hours to produce individual nucleoside monophosphates. Following digestion, add two units of calf intestinal alkaline phosphatase to the samples.
Incubate for an addition 12 hours at 37 degrees Celsius to remove the terminal phosphate groups from the nucleoside monophosphates, and obtain the nucleosides. Prepare a 100-micromolar stock solution of all the modified cytosine nucleosides, and the normal DNA bases in HPLC-grade water for the development of the LC-MS/MS method. Optimize the nucleoside-dependent MS/MS parameters by infusing stock solutions one at a time in the mass spectrometer at a flow rate of 10 microliters per minute in enhanced MS scan mode.
Several parameters can be optimized using the automated quantitative optimization feature of the software for each DNA nucleoside. At this point, optimize the remaining parameters for each DNA nucleoside using the manual quantitative optimization feature of the software in flow injection analysis mode. Now, optimize source-dependent MS/MS parameters by injecting 10 microliters of stock solution using a gradient with 25%solvent B at a flow rate of 0.3 milliliters per minute.
To separate all eight DNA nucleosides, perform liquid chromatography as detailed in the text protocol on a C18 column. Finally, detect and quantify all nucleosides using the LC-MS/MS method in standard curves. Since the catalytic domain of TET2 has a relatively high isoelectric point as compared with most indigenous E.coli proteins, an efficient purification process utilizing a cation exchange chromatography was developed.
This purification yielded greater than 90%pure TET2 enzyme in a single step. In order to separate and quantify different deoxycytidine derivatives and the four natural DNA bases following the TET2 enzymatic reaction, a sensitive LC-MS/MS-based assay was optimized. Standard curves were drawn using serial dilutions of a mixture containing all nucleosides.
The results of the liquid chromatography MS/MS method are shown here. The method allows for separation and quantification of the four normal DNA bases, as well as the four modified cytosine bases. In this experimental procedure, we describe the cloning of untagged human TET2 dioxynase catalytic domain using site-specific recombination technique and a sufficient expression in a destination vector in E.Coli.
We efficiently purified untagged TET2 enzyme utilizing cation exchange chromatography to yield greater than 90%purity in a single step. Further, we have developed a novel liquid chromatography method to separate the four normal DNA bases and the four modified cytosine bases. To quantify the eight nucleosides from TET2 catalyzed reactions, we have coupled our improved liquid chromatography method with tandem mass spectrometry.
The sensitive LC-MS/MS assay was then utilized to determine the activity of recombinant untagged human TET2 enzyme. The approach described here will greatly enhance the valuation of wild-type and mutant TET2 dioxygenase.
