The overall goal of this procedure is to prepare large oligomeric, DNA binding proteins such as MGM 1 0 1 using the maltose binding protein or MVP tagging strategy. This is accomplished by first cloning the MGM 1 0 1 open reading frame downstream of MVP in the PMLC two E vector for expression in e coli. The M-V-P-M-G-M 1 0 1 fusion protein is then purified from e coli by AMLO Affinity chromatography.
MGM 1 0 1 is then released from MVP by Proteolytic Cleavage and separated from MVP by cation exchange chromatography. Finally, the MGM 1 0 1 rings are purified to homogeneity by size exclusion chromatography. Ultimately approximately 0.8 milligrams of soluble MGM 1 0 1.
From one liter of bacterial culture can be used for biochemical and structural characterization of this protein, which is important for DNA repair in mitochondria. The main advantage of using a mortal binding protein tagged version of MGM 1 0 1 is that this version is less toxic, more soluble, and more stable, then the non-tech version. As a result, we can obtain a high level expression of the native MGM 1 0 1 protein in e coli.
Hello, my name is Sarah Vicki. I'm a senior research support specialist in Dr.Jji TEN'S lab here at SUNY Upstate Medical University. My colleague, wan Wong and myself will now demonstrate the expression and purification of MGM 1 0 1 Begin generating the fusion protein by using PCR to amplify the mature MGM 1 0 1 coating sequence.
Do not include the first 22 amino terminal residues of MGM 1 0 1, which are known to be cleaved. After importing into mitochondria, the amplified MGM 1 0 1 is then placed downstream of the MAL E sequence, which encodes the maltose binding protein or MVP in a modified version of the expression vector PM MAL C two E.This generates the M-V-P-M-G-M 1 0 1 fusion with a linker containing a cleavage site for the precision protease. The plasmid is first constructed by selecting e coli transformants without the blue white selection.
The resulting plasmid PLC two E, MGM 1 0 1 is then introduced into the e coli strain, BL two one codon plus DE three RIL, and grown on LB plates that contain ampicillin and chloramphenicol. Begin by inoculating a fresh 10 milliliters of supplemented LB medium with the e coli transformants incubated 37 degrees Celsius overnight with shaking at 200 RPM. The next day inoculate two liters of the supplemented LB medium with 10 milliliters of the pre culture.
Grow the cells at 37 degrees Celsius with shaking. When the OD 600 reaches 0.5, induce expression of the M-B-P-M-G-M 1 0 1 fusion protein by adding IPTG at a final concentration of 0.3 Millimolar grow the cells with shaking at 30 degrees Celsius for five hours. After five hours have passed, transfer the cultures to 500 milliliter centrifuge bottles collect the cells by centrifugation.
After discarding the supernatant add 30 milliliters of lysis buffer containing protease inhibitors and resuspend the cell pellet by pipetting up and down. Next, sonicate the cell suspension on ice for 20 seconds. Using an ultrasonic processor at 50%duty cycle, allow the cells to cool on ice for 30 seconds.
Then repeat the sonication and cooling steps two additional times. Keeping the suspension on ice. Add one milliliter of a two milligrams per milliliter.
DNAs one stock rock the cell lysate at four degrees Celsius for two hours. After two hours have passed. Adjust the final salt concentration to 500 millimolar by adding four milliliters of five molar sodium chloride to 36 milliliters of lysate.
Then to remove the cell debris centrifuge 10, 000 times G at four degrees Celsius for 30 minutes. Transfer the supernatant to a fresh tube. At this point, a 20 microliter aliquot can be used to monitor the efficiency of lysis by sonication.
On an SDS page gel. Next, the cell lysate is passed on an amylose column. To purify the MVP MGM 1 0 1 fusion protein begin by adding 1.5 milliliters of a 50%slurry of AMLO resin to a 15 milliliter conical tube.
Then to equilibrate it, add 13 milliliters of lysis, buffer, and centrifuge at 2000 times G for five minutes. Repeat this wash four more times, then resuspend the resin in 0.75 milliliters of lysis buffer. Add the equilibrated AMLO slurry to the cell lysate rock gently at four degrees Celsius overnight.
The next day, load the lysate amylose resin mix onto an econo column chromatography column installed in a cold room. Open the stop cock to allow the unbound proteins to pass into a collection tube via gravity filtration. Next, wash the AMLO resin by adding 300 milliliters of wash buffer one.
Once it is passed through. Repeat the wash with 150 milliliters of wash buffer two. After adding Ellucian buffer and collecting the EIT as described in the text protocol, determine the yield and purity of M-V-P-M-G-M 1 0 1 by loading an aliquot of the EIT on an SDS page gel.
After the gel has run perform kumasi staining, a major single band of approximately 70 kilodaltons should be visible. Add 50 units of precision protease to the M-V-P-M-G-M 1 0 1 EITs and place in a dialysis tube with dialysis buffer. Allow the cleavage to proceed overnight at four degrees Celsius.
Run an SDS page gel as before. To assess the efficiency of cleavage, a complete cleavage should give rise to two bands of 42 and 28 kilodaltons, which correspond to MVP and MGM 1 0 1 respectively. Load the cleaved MVP MGM 1 0 1 by multiple injections on a bio scale mini macro prep high S cartridge connected to a BioRad biologic DUO flow FPLC system.
Start the cation exchange chromatography by applying a step salt gradient of five millimolar 300, millimolar 500 millimolar 750 millimolar, and 1000 millimolar of sodium chloride. That is created by mixing buffer A and buffer B.Set the flow rate at 0.5 milliliters per minute. Collect two milliliter fractions in a fraction collector, typically MGM 1 0 1 elutes at 500 millimolar of salt.
Combine the fractions for the MGM 1 0 1 peak and check the purity of the protein on an SDS page gel. The MGM 1 0 1 peak should show a single band of approximately 28 kilodaltons after combining the protein fractions from cation exchange chromatography dialyzed the protein in GF equilibration buffer at four degrees celsius overnight. The next, transfer the protein to Aveva spin 15 R ultra filtration spin column and spin at 3000 times G at four degrees Celsius to concentrate the protein.
Once the volume is reduced to approximately one milliliter load MGM 1 0 1 on a calibrated supra six prep grade column equated with the chromatography buffer, start the size exclusion with the chromatography buffer run at a flow rate of 0.5 milliliters per minute. Large oligomeric proteins elute in early fractions, whereas small proteins elute in late fractions. The MGM 1 0 1 rings elute at approximately 400 Kilodaltons.
Collect the fractions of the purified MGM 1 0 1 peaks using a fraction collector load aliquots of the fractions on a 12%SDS page gel in order to check the final quality of the protein. Next, concentrate the protein with Aveva. Spin six by centrifugation at 3000 times G at four degrees Celsius.
Then quick freeze the protein aliquots in micro centrifuge tubes by submerging them in liquid nitrogen for two minutes. Store the samples at negative 80 degrees Celsius within six to 12 months. Use the MGM 1 0 1 samples for single stranded DNA binding assay and for structural visualization by transmission electron microscopy.
The results of the electrophoretic mobility shift assay demonstrates that the purified MGM 1 0 1 has a robust binding activity to single-stranded DNA. The single-stranded DNA probe is increasingly retained in the wells of a native poly acrylamide gel by the Oligomeric. MGM 1 0 1.
As M GM 1 0 1 is added at a concentration greater than 400 nanomolar. These electron micrographs show the higher order structural organization of MGM 1 0 1. The freshly prepared M GM 1 0 1 exists as distinct rings with a diameter of 20 nanometers, which can be revealed by negative stain transmission microscopy.
After several weeks of storage at four degrees Celsius. MGM 1 0 1 forms supramolecular helical filaments Once mastered. This technique can be completed within one week.
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