Figure 4
From: Robust reconstitution of active cell-cycle control complexes from co-expressed proteins in bacteria
Comparison of the kinase activity of CDKA;1-CYCD3;1 and CDKB2;2-CYCB1;2. Upper row, the kinase activity of CDKA; 1-CYCD3;1 and CDKB2;2-CYCB1;2 were normalized against Histone H1. Complexes binding to a Ni-NTA column (His-tagged proteins) were purified from cells either expressing HisGST-CYCD3;1 together with StrepIII-CDKA;1 and Cak1, or HisMBP-CYCB1;2 together with StrepIII-CDKB2;2 and Cak1. Lower row, kinase assays against KRP6 with the kinases as indiated above. Proteins were subjected to SDS-PAGE after the kinase reaction and stained with coomassie brilliant blue (CBB) demonstrating equal protein loading of the substrate. Abbreviations: CBB Coomassie brilliant blue. p-Histone H1 and p-KRP6 for radio-labeled Histone H1 and KRP6, respectively, resulting from kinase assays with radio-labeled ATP.