In order to purify the Mex B membrane protein, Mex B is first expressed in e coli using recombinant DNA methods. Then the membranes are isolated and the membrane proteins are solubilized with a mild detergent. The Solubilized membrane protein is purified through iMac and the protein is further purified through gel filtration chromatography.
The level of purification of the protein is determined using SDS poly ACRYLAMIDE Gel electrophoresis. Though this procedure can provide insight into multi-drug resistance transmembrane transporters, it can also be applied to other membrane proteins demonstrating the procedures today will be formed by a graduate student from my laboratory For this procedure, Mex B from pseudomonas Aosa will sub cloned into the PET 30 B plus expression vector so that Mex B is expressed with a C terminal HEXA histamine tag. Start in the evening by inoculating four three milliliter lb can mycin cultures with fresh transformants or use a frozen stock, grow the cultures on a roller at 37 degrees Celsius overnight in the morning.
Use the overnight cultures to inoculate 150 milliliters of LB containing 30 micrograms per milliliter. Can Mycin grow the culture at 37 degrees Celsius on a shaker in the afternoon? Use the small culture to inoculate six times one liter, two XYT media containing 30 micrograms per milliliter.
Can mycin in fern back flasks? Use 25 milliliters per culture for a one to 40 dilution. Grow the cultures at 37 degrees Celsius until they reach an optical density.
600 of 0.4 to 0.6, about 1.5 hours. When the cultures reach the proper density, induce protein expression by adding 0.5 milliliters of one molar.IPTG. Put all the flasks back in the shaker and continue to grow them at 30 degrees Celsius overnight until harvesting the bacteria the following morning.
Retrieve the cultures from the shaker and chill them. Then to harvest the cells, transfer the cultures to centrifuge tubes and centrifuge at four degrees Celsius for 30 minutes at 5, 000 rotations per minute in a large scale centrifuge while the cells are centrifuging. Supplement 100 milliliters of cell re suspension buffer with DNAs protease inhibitors and lysozyme.
Also supplement two aliquots of membrane Resus suspension Buffer with protease inhibitors as shown in this table. Keep all three solutions on ice when centrifugation is complete. Resus spend the cell pellets in 100 milliliters of cell resus suspension Buffer.
Next, extract the cells. The cell suspension is loaded into the French pressure cell and the cell is placed on the French press. The pressure is applied until it reaches 12, 000 pounds per square inch.
The valve is opened a very small amount so that the broken cells trickle out. It is important not to open the valve too much and let the cells spurt out because they will be released with too little pressure and will not be efficiently broken. Collect the cell lysate in a bottle, kept cold on ice.
Pass the cell solution through a French pressure cell again at 12, 000 pounds per square inch. Transfer the cell lysate to SS 34 centrifuge tubes and centrifuge to remove cell debris at 10, 000 rotations per minute for 30 minutes at four degrees Celsius in an SS 34 rotor. The clarified lysate is next.
Used to prepare the membrane fraction. Carefully transfer the supinate into ti. 6 4, 7 0.5 ultra centrifuge tubes, centrifuge in a TI 6 4 7 0.5 rotor at 40, 000 rotations per minute for 50 minutes at four degrees Celsius.
Discard the supinate. Use a pipette to gently resuspend the pellet, which contains the cell membranes in approximately 25 milliliters of membrane Resus suspension buffer transfer the membrane suspension to a clean centrifuge tube and centrifuge gain in a TI I 6 4 7 0.5 rotor of 40, 000 rotations per minute for 50 minutes of four degrees Celsius and resuspend the washed membrane palette in 25 milliliters of membrane Resus suspension buffer. Next, solubilize the membrane proteins to the resuspended membranes at about 25 milliliters.
Add six milliliters of 10%DDM so that the final detergent concentration is 2%DDM rock the mixture at four degrees Celsius for two hours after the two hour incubation in the presence of DDM centrifuge, the mixture at 40, 000 rotations per minute for 40 minutes at four degrees Celsius in the TI I 6 4 7 0.5 rotor. In order to separate the soluble protein detergent complexes from the insoluble proteins. Save the supine natant that contains the Mex B protein detergent complexes to use in the purification mix the supine natant containing the Mex B protein detergent complexes with two milliliters of the lon metal affinity beads equilibrated in Resus suspension Buffer incubate for one hour on a roller at four degrees Celsius after binding of the proteins to the resin.
Pour the slurry into a gravity flow column body and discard the flow through. Wash the column with 20 milliliters or 10 column volumes of iMac binding and wash buffer when washing of the column is complete. Elute the bound proteins with 10 milliliters of iMac elution buffer.
Take 10 microliter samples of each elution fraction. Mix with 10 microliters of two times SDS sample, buffer, and analyze on a 10%poly acrylamide. SDS gel.
Estimate the amount and purity of mex B in each fraction. Pull the fractions containing the Mex B protein detergent complexes and concentrate them in a spin concentrator at four degrees Celsius. Be careful that the protein does not precipitate out at this step.
Use several short spins and watch for precipitation. After each spin, then proceed to purify the pooled fractions using a gel filtration column. Pre equilibrate a super rose 12 HL 30 over 10 column with 24 milliliters of running buffer and wait for a flat baseline.
Next, rinse the actor system loading loop with running buffer. Before applying the protein to the column, filter the protein solution using a syringe filter. Then load up to 240 microliters of the protein solution onto the column with a protein of up to five milligrams per milliliter.
Run 1.5 column volumes of buffer and collect 0.25 milliliter fractions. The XB protein detergent complexes elute as a peak at around 10 to 15 milliliters of elution volume. Take five microliter samples of the peak fractions.
Mix each sample one to one with two times SDS sample buffer. Analyze the samples on a 10%poly acrylamide gel to estimate the amount and purity of me Mex B in each fraction, pull the fractions containing pure ME B.This poly acrylamide gel was loaded with pulled fractions from the iMac column and individual fractions from the gel filtration column. After the gel filtration column, the protein appears pure on the kumasi stained poly acrylamide gel.
A trace from the gel filtration column shows the main peak of the protein detergent complex eluting from the column. The average yield of mxb protein is approximately two milligrams per six liters of two XYT culture Once mastered, this procedure can be done in eight hours if it is performed properly. After watching this video, you should have a good understanding of how to express solubilize and purify a membrane protein.
