This method can be used not only to address key questions in basic research, but also contribute to the medical, pharmaceutical, and biotechnological fields, by delineating novel small molecular ligands of essential proteins. The main advantage of this technique, is that it enables parallel analysis of protein and metabolic partners of the protein of choice in closely vivo conditions. This method helps to fish out interacting molecules without being restricted to pre-selected ligands.
Though this method was developed for plants, it can also be applied for other systems, such as HeLa cells, E.coli, and S.Cerevisiae. To begin, prepare MSMO medium and adjust the pH to 5.7 with one molar potassium hydroxide. Then, autoclave the solution for 15 minutes at 121 degrees Celsius.
Add the supplements to the medium just before the start of the experiment. Now, grow transgenic PSBL Arabidopsis thaliana cell culture in 50 milliliters of MSMO medium in a 100 milliliter flask. Then, place the flask on an orbital platform shaker and gently agitate the cells at 130 rpm at 20 degrees Celsius in the light.
Every seven days, subculture the cells in fresh medium at a one to 10 solution. Next, use a glass funnel with a nylon mesh fitted on a conical flask, connected to a vacuum pump to harvest the cells during the logarithmic growth phase. Then, wrap the dried cells in aluminum foil, and freeze in liquid nitrogen.
Using a pre-chilled mixer mill, set at a vibrational frequency of 20 hertz. Homogenize the harvested and frozen plant cell material to a fine powder for two minutes. Next, aliquot three grams of the ground powder per sample using liquid nitrogen, pre-chilled equipment to prevent it from thawing.
Then, using a liquid nitrogen pre-cooled mortar, triturate the ground powder with three milliliters of ice cold lysis buffer until it thaws. Divide the thawed sample immediately into two milliliter micro-centrifuge tubes. Centrifuge the samples at 20, 817 g for 10 minutes at four degrees Celsius to remove the cellular debris.
While the sample is being centrifuged, aliquot 100 microliters of IgG sepharose beads per sample. Add one milliliter of lysis buffer, and re-suspend the beads by vortexing. Flash spin for three seconds at 2, 000 g, and discard the lysis buffer.
Finally, re-suspend the beads in 400 microliters of lysis buffer. After centrifugation, transfer three milliliters of the clear plant lysate to a 15 milliliter conical centrifuge tube. Add the pre-equilibrated beads to the plant lysate, and incubate the mixture on a rotating wheel for one hour at four degrees Celsius.
Transfer the mixture into a syringe connected to a lure locked cap on a spin column with a 35 micrometer pore sized filter located on top of the vacuum manifold. Pass the lysate through the unit by turning on the vacuum pump with gentle pressure to avoid harm to the beads. The unbound lysate is drained into the waste station located at the base in the manifold and the protein complexes, bound to the beads, remain on the filter.
Once the syringe is emptied off the cell lysate, add 10 milliliters of wash buffer to wash the beads. Then, add on milliliter of elution buffer to perform the second washing step. Now, remove the syringe and lure lock cap.
Close the bottom cap at the column, and add 400 microliters of the elution buffer containing 50 units of the enhanced form of tobacco etch virus, protease, to the beads. Place the column on a table shaker, set at 1, 000 rpm for 30 minutes at 16 degrees Celsius. Then, add additional 50 units of the protease to the column, and incubate the mixture on the shaker as in the previous step.
Next, remove the bottom cap, and replace the column in a two milliliter micro-centrifuge tube. Centrifuge at 20, 817 g for one minute to collect the eluate and proceed with protein and metabolite extraction. To begin the extraction, add one milliliter of three to one to one MTBE methanol water solvent to the eluate.
Invert the tube to mix to the sample. Next, add 0.4 milliliters of one to three methanol water solution to the mixture, and invert the tube to mix the sample. Then, centrifuge the sample at 20, 817 g for two minutes at room temperature to allow phase separation.
Using a one milliliter manual liquid handling pipette, remove the upper phase containing lipids. Then, add 0.2 milliliters of methanol, and invert the tube to mix the sample. Centrifuge at 20, 817 g for two minutes at room temperature, and collect the polar phase in a new tube for metabolite measurements.
Leave approximately 50 microliters of the polar phase at the bottom of the tube to avoid dislodging the protein pellet. Next, dry the tube containing the polar phase in a centrifugal evaporator over night. Dry the tubes of protein pellet for 30 to 60 minutes to avoid over drying.
Using one-step affinity purification, together with mass spectrometry protein-protein and protein-metabolite interactions in transgenic arabidopsis thaliana cell cultures have been identified. Cell cultures expressing NDPK1, show significant enrichment in valine-leucine, isoleucine glutamate, leucine isoleucine, and isoleucine phenylalanine when compared to empty vector, NDPK2 and NDPK3 samples. To look for known protein-protein and protein-metabolite interactions, 13 proteins and four dipeptides co-eluting with NDPK1, are queered against the stitch database.
The main associations are seen between APX1 ortholog and the aldehyde dehydrogenase family, and the translation initiation factor, FBR12, with translation initiation factor two subunit alpha homolog. The identified dipeptides show no reported protein partners. Once mastered, this technique can be done in four hours, if performed properly.
While attempting this procedure, it is important to keep samples, equipment, and reagents cold throughout the experiment. Following this procedure, other methods, like microscope thermophoresis or activity assay can be performed in order to determine direct interaction between molecules or inference of their ligand on protein activity. After watching this video, you should have a good understanding of how to handle the sample during the whole procedure to obtain trustworthy results and identify ligands of your protein of choice.
Don't forget that working with methanol and MTBE solution can be extremely hazardous. Appropriate personal protective equipment should be worn, and precautions, such as working under the fume hood, should always be taken while performing this procedure.
