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The aim of this experiment is to identify protein, protein interactions and the subunit composition of native MultiPro complexes from e coli. This is achieved by amplifying the sequence specific linear PCR products, encoding the SPA tag and a selectable marker, which are integrated and expressed in frame as carboxy terminal fusions in a ddy 3, 3 0 background using bacteria Fage lambda recombination system as a second step dual step purification is performed using cow modlin and Anti-Flag affinity beads to efficiently recover low abundance protein complexes. Next, the bound proteins alluded with cow modlin, elution buffer or CEB are digested with trypsin and processed with mass spectrometry for protein identification.

Results obtained show that the several subunits of the core RNA polymerase enzyme, including the transcription termination, anti determination factors co purified efficiently with tagged RNA polymerase subunit D protein, indicating that the newly associated components can be efficiently uncovered using this approach. Generally, individuals new to this method may struggle with two round affinity purifications, where careful processing of the cell lysates with appropriate buffer solutions is absolutely necessary to ensure success in efficient recovery of low and high abundance burden complexes from large scale cultures Demonstrating the affinity purification and mass spectrometry procedures will be Olga Kagan and HBO guo two technicians in my laboratory. This protocol begins with the targeting of specific amplified sequences to the DDY three 30 strain in which the Lambda red recombination machinery is expressed as described in the written protocol.

Next, where the bacteria farge Lambda recombination system expressing strain overnight in two milliliters of Luria bati or LB medium at 32 degrees Celsius, shaking at 180 RPM the following day, inoculate one milliliter of the overnight culture into 70 milliliters of fresh LB medium in a 500 milliliter conical flask. Grow the inoculum at 32 degrees Celsius by shaking at 180 RPM until the OD 600 reaches about 0.8. Induce the cells by incubating the flask in a water bath at 42 degrees Celsius by gently shaking at 180 RPM for 15 minutes.

Immediately after the induction, incubate the flask in an ice water slurry bath for at least 30 minutes with shaking. After harvesting and washing the cells as described in the written procedure, Reese suspend the cell pellet in 700 microliters of ice, cold, sterile water resulting in electro competent cells through electroporation. Introduce one microliter of the purified amplicon into 40 microliters of the electro competent cells.

The cells are electroporated after homologous recombination and integration. The transformants that successfully recombined the tag slash cassette into the chromosome are selected based on resistance to can mycin select multiple transformers for western blotting to verify the correct generation of the SPA tagged fusion proteins with Anti-Flag M two antibody that is selective against the flag epitopes of the SPA tag Transfer 10 milliliters of the overnight culture into 990 milliliters of fresh tb. Supplemented with 25 micrograms per milliliter of can mycin in a four liter flask.

Grow the culture at 32 degrees Celsius with constant shaking at 250 RPM for five to six hours until the OD 600 reaches between two to three. Transfer the one liter e coli SPA tag culture to clean centrifugation bottles and spin the cells at four degrees Celsius and 3, 993 times G for 15 minutes. After resus suspending the cells as described in the written protocol.

Transfer the samples to a sterile stainless steel cup placed on ice for sonication. Submerge the probe into the sample and sonicate for three minutes. Following sonication, allow an additional two minutes to cool the sample from overheating following centrifugation of the sonicate lysate.

Carefully transfer the S supernatant from the centrifugation tube to a 50 milliliter polypropylene falcon tube. Freeze the sample using liquid nitrogen and store the sonicated frozen cell extract for a maximum period of six months at minus 80 degrees Celsius for future use. To begin affinity purification for the frozen sonicated cell extract by placing the tube in cold water, incubate the TH cell extract with three microliters of benzo, a nuclease for 30 minutes of four degrees Celsius.

To this mixture, add non ionic detergent Triton X 100 and 200 microliter suspensions of Anti-Flag M two agro beads. Gently mix the content by rotating the tube for three hours at four degrees celsius after three hours of rotation, centrifuge the tube at 1, 700 times G for six minutes. Following centrifugation carefully remove as much of the sate as possible without disturbing the loose speed pellet.

Re suspend the pellet in the remaining SNA and then transfer into a polypropylene prep column. Remove the bottom outlet plugs of the column to allow the eluate to drain by gravity.Flow. Next, wash the column five times with 200 microliters of one times a FC buffer and proceed to Cleve with TEV as described in the written protocol.

Following cleavage fast on both the top and the bottom of the column tightly. Gently mix the contents in the column by rotating overnight at four degrees Celsius after overnight rotation. Drain the eluate by removing the top cap and the bottom plug into the new column.

Wash the old column with 400 microliters of one x cal Modlin binding buffer and 150 microliter suspension of Cal Modlin binding beads. Elute the bound protein in four fractions of 50 microliters in a fresh eend orph tube. Using one x cal Modlin elution buffer.

Distribute the eluted fraction into two clean eend orph tubes in equal volumes. Then dry down the contents of both tubes using a speed vacuum. The dried eluate from one tube is used for running a silver staining gel as described in the text while the other is stored in minus 80 degrees Celsius.

For future use in mass spectrometry to the dried sample, add 50 microliters of the digestion buffer and 0.9 microliters of 100 millimolar tris phosphine hydrochloric acid incubate the mixture for 45 minutes of room temperature for the reduction step Next, add one microliter of the 500 millimolar IDO acetamide and incubate in the dark for another 40 minutes. To allow for sample alkylation After the second round of incubation, add one microgram of trips into the mixture. Incubate the sample either at 37 degrees Celsius for five hours or overnight at room temperature following incubation.

Be sure to stop the reaction by adding one microliter of acetic acid. Then prepare the Millipore zip tip, pipette tip as described in the text for efficient binding of the peptide to the tip pipe pep. Mix the peptide mixture 20 times wash off the peptide mixture that it did to the tip by aspirating and dispensing the washing solution.

Repeat this procedure twice for efficien binding of the peptide mixture to the tip with the tip containing the bound peptide. Aspirate 10 microliters of the wetting and e equilibration solution and dispense into a clean eend orph tube. Repeat this step twice.

After drying the eluted samples in a speed vacuum, the samples can be analyzed immediately by mass spectrometry or stored at minus 80 degrees Celsius prior to use. The micro columns used in this procedure are packed with approximately 10 centimeters of three micron lunar C 18 resin and are interfaced to a pro Zion nano electrospray ion source that is placed in line with the orbitrap instrument. A pro Zion nano flow binary HPLC pump is used to deliver a stable tip flow rate of approximately 300 nanoliters per minute during the peptide separations to achieve peptide dilution set up an organic buffer gradient according to the sample complexity.

For this e coli SPA sample, the mobile phase solvent A has 95%HPLC gradient water and 5%aceto nitrile with 0.1%formic acid while solvent B has 5%HPLC gradient water and 95%aceto nitrile with 0.1%formic acid following mass spectrometry search the spectra using a database search algorithm such as seaquest against a database of e coli protein sequences. Statistically filter the result using a probability algorithm such as star quest to ensure a low false discovery rate, both SPA tagged RNA polymerase sigma factor and yak LA protein of unknown function co purified specifically with the core RNA polymerase enzyme including alpha beta and beta prime subunits, as well as the RNA polymerase recycling factor. In contrast to tagged RNA polymerase sigma factor yak L bound additionally to an essential transcription termination anti determination factor suggesting a specialized function in transcription.

Several other smaller co purifying proteins were were also detected by LCMS that were not apparent on the gel, including the RNA polymerase Omega subunit and transcription, termination and determination factors and USA and NUSD. Conversely, in an independent experiment tagged mobilization of sulfur machinery, S-U-F-B-S-U-F-C and SUFD co purified with each other, indicating joint participation in iron sulfur cluster biosynthesis as a single scaffolding complex. This representative example highlights the fact that previously well-studied highly annotated bacterial MultiPro complexes participating in essential biological processes often have novel associated components that can be efficiently identified.

Using this approach, the low copy number of the SUFB protein may have resulted in weak signal intensity relative to the strong intensity observed from the SUFC and SUFD pull down experiments to define the composition of stable multi-unit protein complexes, co purification scores were assigned to high confidence interactions by taking into account the uniqueness of bait prey, bait bait and prey prey relationships. Then using graph clustering procedures like the markoff clustering algorithm. Discrete protein clusters are identified from the partitioned probabilistic PPI network.

Putative interaction networks can be visualized using cyto scape. Combining proteomics data with additional functional association evidence inferred by genomics methods can be used to investigate novel mechanistic roles of previously annotated e coli proteins. Here, subnets of protein complexes and functional modules that participate as broader functional neighborhoods have been defined in e coli.

The main hierarchical cluster Graham shows the patterns of functional predictions and existing annotations for the functionally orphan and characterized genes of e coli, yellow and blue colors represent existing and inferred functions respectively while shade intensities reflect confidence scores. Biological processes associated with various neighborhoods are indicated on the right insets show the individual components of a representative neighborhood based on the integrated physical and functional interaction network similarity scores. After watching this video, you should have a good understanding of how stable protein complexes from estia Coli can be isolated using the affinity purification coupled with mass spectrometry approach.

In principle, this technique can be applied to characterize membrane protein complexes or protein complexes from any other species for it recombining as possible.