The overall goal of the following experiment is to investigate ligamentization propensities of protein, transmembrane domains or T MDs in a natural membrane environment. This is achieved by expressing the transmembrane domain of interest in e coli as a chimeric protein with maltose binding protein for localization to the peri plasm and tox. R as a transcriptional reporter, transmembrane domain induced ligamentization results in dimerization of tox R and activation of LXi transcription As a second step cells are lysed and treated with ONPG, which is hydrolyzed by beta galacto to the light absorbing species O nitro pholate or ONP.
Next ONP levels are quantified by measuring absorbence of light at 405 nanometers. In order to determine the levels of transmembrane domain, ligamentization results reveal the ligamentization propensity of the transmembrane domains based on a tox R reporter assay. The main advantage of this technique over existing biophysical methods such as SDS page or fluorescence, is that the behavior of the transmembrane domain is studied in a natural phospholipid bilayer by expression in e coli rather than a membrane mimetic system such as a detergent myself.
This method can help answer key questions in the membrane protein field, such as elucidation of key structural features involved in transmembrane Helix Association Prior to the start of this protocol pox seven plasmids expressing the required chimeric protein are prepared from commercially synthesized oligonucleotides, representing the TDS of interest flanked by NHE one and BMH one. Restriction sites after plasmids have been prepared gently thaw fhk 12 competent cells on ice. Once thawed transferred the cells into 15 milliliter culture tubes at 200 nanograms of each plasma DNA to a separate tube and incubate the cells on ice for 30 minutes.
Then heat shock the cells for 90 seconds at 42 degrees Celsius, followed by incubation on ice for two minutes at 800 microliters of SOC media and incubate the samples at 37 degrees Celsius with shaking for one hour after the incubation inoculate five milliliters of LB media containing chloramphenicol and aose with 50 microliters of the transformation mixture using 15 milliliter culture tubes in triplicate for each sample. Finally, incubate the samples at 37 degrees Celsius with shaking for 20 hours. To measure the all ligamentization of TDS beta galacto tase activity is determined.
First, preheat the plate reader to 28 degrees Celsius. Use Z buffer stock to prepare fresh Z buffer solutions as described in the written protocol. Transfer Z buffer chloroform from the tube to the reservoir, making sure to only pipee the upper aqueous layer of the Z buffer chloroform.
Place the 96 well plate on a piece of paper with a grid drawn to ensure accurate pipetting transfer 100 microliters of Z buffer chloroform to each well of a 96 well plate, followed by five microliters of each culture in quad. Duplicate omit the culture from the four wells that will serve as blanks. Measure the OD 5 95 of the plate to determine cell density.
Add 50 microliters of Z buffer SDS to all wells of the plate. Then shake the plate in the plate reader for 10 minutes to ly the cells following cell lysis at 50 microliters of ZPA for ONPG to all wells. Return the plate to the plate reader and measure OD 4 0 5 every 30 seconds for 20 minutes.
Determine the level of beta lacto sase activity through a calculation of Miller units normalizing to the blank. Western blotting is performed to verify equivalent protein expression between constructs. First, combine 50 microliters of the triplicate cultures and then centrifuge the samples using a micro centrifuge.
Remove the supernatant by pipetting and resus. Suspend the residual pellet in two x sample loading buffer load 7.5 microliters of each sample on a standard 8%gel and carry out electrophoresis at 125 volts for one hour and five minutes after the transfer. Incubate the membrane with anti M-V-P-H-R-P conjugated antibody to visualize the chimeric protein is observed at approximately 70 kilodaltons with some degradation products sometimes seen around 48 kilodaltons.
Endogenous MVP is also observed at 45 kilodaltons. In order to assess proper membrane insertion of the chimeric TMD construct, the maltose binding protein deficient PD 28 cell line is used when grown in minimal media with maltose as the sole carbon source only cells expressing a membrane integral expression product with maltose binding protein correctly located in the peri plasm can grow. Transform PD 28 cells as described for fhk 12 cells and inoculate two milliliters of LB medium.
Grow the cells at 37 degrees Celsius with shaking at 300 RPM overnight after the incubation. Pellet the cells by centrifugation and wash by Resus suspension in two milliliters of PBS and gentle pipetting with a large tip. After pelleting, the cells again wash with PBS for a second time, then perform a final centrifugation and resus suspension in one milliliter of PBS.
Use 25 microliters of the resuspended cells to inoculate five milliliters of minimal media. Incubate the cells of 37 degrees Celsius with shaking at 15 hours. Transfer 200 microliters of each sample into a 96 well plate, and take the OD 5 9 5 reading using the plate reader.
Repeat this process every two hours until 25 hours. The all ligamentization propensity of individual transmembrane domains from the multis spanning membrane integral protein latent membrane protein one was analyzed using the tox R transcriptional reporter assay. Transmembrane domain five exhibits a strong propensity to liga rise as demonstrated by high Miller units, which is comparable to the positive control GPAA well-established dimerizing sequence.
A deleterious mutation in transmembrane domain five D one 50 A reduces the ability of the sequence to all liga rise LMP one, TM one does not significantly all liga rise and exhibits a very low Miller unit signal just above the signal for blank, which is non transformed FHK 12 cells. After watching this video, you should have a good understanding of how to determine if a transmembrane helix of interest has the ability to form higher order complexes in a natural membrane environment While attempting this procedure, it's important to pipette extremely carefully into the 96 well plate to avoid large errors and ensure that no air bubbles are introduced.
