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Reconstitution and use of highly active human CDK1:Cyclin-B:CKS1 complexes

Protein Sci. 2021-11; 
Pim J Huis In 't Veld, Sabine Wohlgemuth, Carolin Koerner, Franziska Müller, Petra Janning, Andrea Musacchio
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Proteins, Expression, Isolation and Analysis After further purification using a Heparin HP column (GE Healthcare), CENP‐T was fluorescently labeled with a GGGGK‐TMR (5‐Carboxytetramethylrhodamine) peptide (GenScript) using the Calcium‐independent Sortase 7?M Get A Quote

摘要

As dividing cells transition into mitosis, hundreds of proteins are phosphorylated by a complex of cyclin-dependent kinase 1 (CDK1) and Cyclin-B, often at multiple sites. CDK1:Cyclin-B phosphorylation patterns alter conformations, interaction partners, and enzymatic activities of target proteins and need to be recapitulated in vitro for the structural and functional characterization of the mitotic protein machinery. This requires a pure and active recombinant kinase complex. The kinase activity of CDK1 critically depends on the phosphorylation of a Threonine residue in its activation loop by a CDK1-activating kinase (CAK). We developed protocols to activate CDK1:Cyclin-B either in vitro with purified CAKs or in... More

關(guān)鍵詞

CDK1, CKS1, cell cycle, cyclin, cyclin dependent kinase, cyclin-B, phosphorylation, phostag, processivity, recombinant protein
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