Cyclin-dependent kinases (CDKs) are the proteins involved in the cell cycle control. Seven types of CDKs named from A to G occur in plants. In Arabidopsis thaliana, the unique CDKF type is represented by only one member called CDKF;1, which is localized in the nucleus and cytoplasm [1]. CDKF;1 was shown to be auto-phosphorylated and to play a superior role in the hierarchy of other CDKs activating kinases [2]. Moreover, the activity of CDKF;1 is cyclin-independent. Knocking out of the Arabidopsis gene encoding CDKF;1 results in severe developmental defects [3]. Although its unique properties, the three-dimensional structure of CDKF;1 is still unknown. Application of cryo-electron microscopy (cryo-EM) for structural studies of CDKF;1 could help to understand the mechanism of its action. AlphaFold predictions [4] and stability tests are consistent that the C-terminus end can hinder obtaining proper CDKF;1 structure. Therefore a truncated version of CDKF;1, with deletion of the last 34 amino acid residues, was prepared. Given that the molecular weight of that CDKF;1 variant is relatively too low for cryo-EM analysis, different approaches to solve this problem are investigated.
The aim of the planned studies was to produce monoclonal antibodies specific to Arabidopsis CDKF;1. These antibodies will be used to prepare a complex of antibody-CDKF;1 with molecular weight high enough to perform cryo-EM analysis. To produce the monoclonal antibodies hybridoma protocol [5] was used. As a result, two different monoclonal antibodies were obtained and their utility for determination of the CDKF;1 protein structure using cryo-EM is currently tested.