Process
There are a few methods in which the fusion protein can be introduced into the host. If the host is yeast, then one of the methods may be the use of plasmids that will eventually translate the fusion protein within the host. Whichever method that is being used, it is preferable to maintain expression of the fusion protein as close as possible to its natural level.
Once the fusion protein is translated within the host, the new protein at one end of the fusion protein would be able to interact with other proteins. Subsequently, the fusion protein is retrieved from the host by breaking the cells and retrieving the fusion protein through affinity selection, together with the other constituents attached to the new protein, by means of an IgG matrix.
After washing, TEV protease is introduced to elute the bound material at the TEV protease cleavage site. This eluate is then incubated with calmodulin-coated beads in the presence of calcium. This second affinity step is required to remove the TEV protease as well as traces of contaminants remaining after the first affinity step. After washing, the eluate is then released with ethylene glycol tetraacetic acid (EGTA).
The native elution, consisting of the new protein and its interacting protein partners as well as CBP, can now be analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or be identified by mass spectrometry.
Read more about this topic: Tandem Affinity Purification
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