The overall goal of this procedure is to identify membrane proteins expressed in SIA coli for structural and functional analyses. First, the bacteria are harvested, lies and analyzed by SDS page for visualization of the correctly folded GFP proteins by Ingel fluorescence. In the second step production of the e coli determined to express the membrane proteins of interest is scaled up and a total membrane fraction is prepared from the cells following the induction of the protein expression.
Finally, the total membrane fraction is solubilized in four different detergents and the mono dispersity of the detergent.Solubilized. GFP proteins is analyzed by size exclusion chromatography. Ultimately, the proteins can be monitored by fluorescence and the elucian profiles plotted to assess the aggregation mono dispersity and free GFP in the samples.
The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to their low solubility and they inherit instability once extracted into detergents. Experience has shown that screening multiple variants of membrane proteins such as orthologs from different species can improve the success rate of membrane protein expression. Fusion to green fluorescent proteins used to report authentic folding.
The first step in our GFP based protocol for the expression screening of membrane proteins and e coli uses ingel fluorescence of detergent lysates from whole cells to identify the membrane protein constructs for further analysis based on the level of expression in the screen, The behavior of selecting membrane proteins and different detergents is then evaluated using fluorescence detection coupled to size exclusion chromatography. This provides information about the mono dispersity of the detergent protein complexes. To analyze IPTG induced bacterial expression begin by transferring one milliliter of IPTG treated e coline duplicate from overnight culture 24 deep well blocks into 96 deep well blocks, seal the blocks and then spin down the cells for 10 minutes at 6, 000 times G at room temperature.
Afterwards, invert the block to decant the media tapping gently on a paper towel to drain any remaining liquid. Seal the plate again and store the cells at minus 80 degrees Celsius after at least 20 minutes. Defrost the pellets for 20 minutes at room temperature and then use a multichannel pipette to resuspend the cells completely in 200 microliters of lysis buffer.
Treat the cells with 20 microliters of DLPI solution was shaking at 1000 RPM and four degrees Celsius for 10 minutes. Then add 25 microliters of 10%doda multiside to each well and incubate the cells for another 60 minutes with shaking. After the DDM treatment centrifuge, the deep well block and then transfer 10 microliters of the cleared lysate to a microtiter plate and add 10 microliters of SDS page gel loading buffer.
Now load the gel with 10 microliters of the lysate buffer solution per well and run the gel at constant voltage between 100 to 120 volts in a cold room for two to 2.5 hours when the diaphragm reaches the bottom. Place the gel onto an imager with a blue light filter to detect the fusion proteins to scale up the cell cultures. After transforming an Eloqua of competent e coli, pick a colony into 10 milliliters of power broth for overnight growth at 37 degrees Celsius.
The next morning dilute five milliliters of the overnight culture into 500 milliliters of power broth and continue to grow the cultures with shaking at 250 RPM and 37 degrees Celsius. When the OD at 595 nanometers is approximately 0.5, induce expression by adding IPTG to a final concentration of one millimolar, and then place the culture back in the shaker overnight this time at 20 degrees Celsius. The next morning, harvest the cells by centrifugation and store the pellets at minus 80 degrees Celsius.
To prepare the cell membranes. Re suspend the third room temperature pellets in five milliliters of PBS per gram of cell pellet, increasing the volume as necessary until the viscosity reduces to a runny consistency. Then add magnesium chloride to a final concentration of one millimolar DNAs to a final concentration of 10 micrograms per milliliter and one prepackaged protease inhibitor tablet per 20 gram of cell pellet to the cells.
Next, break open the cells using a chilled cell disruptor at a pressure of 30 KPSI and then pellet the unbroken cells and debris for 15 minutes at 30, 000 times G and four degrees Celsius. Transfer the supinate to an ultracentrifugation tube and collect the total membrane fraction by spinning down the supinate for one hour at 200, 000 times G and four degrees Celsius. Then use the cell homogenizer to re suspend the membrane pellet in 10 milliliters of ice cold PBS to solubilize the membranes for each detergent to be used.
Aliquot 900 microliters of the membrane suspension into 1.5 milliliter poly centrifi tube. Then add 100 microliters of each freshly prepared detergent to the appropriate 1.5 milliliter tubes and incubate the mixtures at four degrees Celsius with mild vegetation. After an hour, pellet the detergent and soluble fractions with a benchtop ultra centrifuge, ensuring the tubes are at least half full and retain the supine agent for fluorescent detected size exclusion chromatography or FEC analysis.
Next, a equator size exclusion column with a broad fractionation range with running flow buffer at a rate of 0.3 milliliters per minute. Then inject 100 microliters of a solubilized membrane sample onto the column at the same flow rate and monitor the elucian profile using fluorescence optics. Finally, import the elucian volume and fluorescence intensity data into a spreadsheet program for graphical display and analyze the remaining solubilized membrane material by ingel fluorescence.
As demonstrated earlier, the said family of proteins is essential for bacterial cell division. In these images, a representative in gel fluorescence visualization of 24 OUTTA 47 said constructs is shown. The intensity of the bands gives an indication of the level of expression.
For example, the fusion protein in lane scene four is expressed well in both strains. However, the additional lower molecular weight bands suggests that the protein is partially degraded in many lanes. There is a band that corresponds in size to GFP alone.
For example, G four and H four are completely broken down to free GFP in the C 41 DE three P lies S strain. Whereas in the LEMO 21 DE three, there is some intact protein in these graphs. Analysis of two constructs that performed well in the primary screen by fluorescence detected size exclusion demonstrates how the proteins behave differently depending on which of the four detergents were tested.
For example, this protein gave only a symmetrical peak with litter aggregation in Dool Multiside making it the detergent of choice for subsequent purification. By contrast, the other fusion protein exhibited a similar profile in all of the detergents tested. This protocol can be readily expanded to include, for example, different e coli strains grown under different expression conditions and additional deterrents in both primary and secondary screens.
In summary, the main purpose of this protocol is to enable a large number of membrane proteins to be screened in terms of expression and detergent ization in order to identify candidates for further analysis.
