Establishment of de novo recombinant CDK11B/Cyclin heterodimers
To develop a de-novo assay for activity screening against the CDK11b kinase. We needed to create a soluble CDK11b construct and find a suitable cyclin partner. Neither a tool compound nor a substrate was available for assay development.
CDK enzymes bind to regulatory proteins called cyclins to coerce their activity. The project focused on the identification of suitable assay components:
- A construct for expression of soluble CDK11b
- A suitable cyclin partner for CDK11b
- Purification tags for both CDK11b and the cyclin partner
- A substrate to determine the enzymatic activity of the CDK11b/Cyclin complex
- Optimization for optimal assay window and reproducibility
We created various CDK11b plasmids in the first step, which were co-expressed in insect cells with a potential cyclin partner. We found one construct which resulted in a soluble CDK11b/cyclin protein complex. In a second step, we tried to detect the enzymatic activity of the new construct with our Substrate Finder. Unfortunately, none of our common generic substrates were suitable. Literature research pointed us towards a potential substrate, which was successfully phosphorylated by our CDK11b construct. To increase the CDK11b activity, we co-expressed it with several other cyclins and identified Cyclin K as the best partner. Careful titration of all assay components and testing of various tags for both CDK11b and Cyclin K resulted in significantly increased CDK11b activity by 20 fold resulting in a reliable assay with high reproducibility and an excellent noise-to-signal ratio.