CDKL5 disorder is a neurodevelopmental disorder affecting children. Its symptoms include siezures and neurological deficits, similar to Rett syndrome.
The mechanisms of CDKL5 disorder aren't understood well. It remains unclear how CDKL5 interacts in the cell to cause such strong symptoms. As a result, it has been difficult to develop therapies for patients affected with the disorder.
The purpose of this project was to identify proteins that CDKL5 interacts with in the brain. By identifying interactors, we can better understand the molecular mechanisms behind CDKL5 disorder.
One of the ways to do this is to isolate CDKL5 protein and its interactors from other cell components, and analyze them using a technique called mass spectrometry. My goal this summer was to properly isolate the CDKL5 protein and its interactors.
In order to do this, I used a mouse that has a tag attached to its Cdkl5 gene. This tag allows for the protein to be isolated, due to its strong chemical interactions. However, while isolating the CDKL5 protein itself was effective, I had difficulty isolating the interacting proteins. Thus, I optimized the protocol to be more efficient at isolating CDKL5 and its interactors. To do this, I altered the biochemistry of the reagents I was using. For example, I adjusted concentrations of salinity, pH, and the strength of the chemical interactions in the protocol.
As a result, I was able to create a protocol to isolate CDKL5 and its interactors from the cell. The next step is to analyze tese interactors using mass spectrometry, and identify which proteins are the most essential. This will help us garner an understanding of the disease and be the baseline for therapeutic development.