Figure 2
From: Robust reconstitution of active cell-cycle control complexes from co-expressed proteins in bacteria
Assessment of CDKA;1-CYCD3;1 activities from proteins purified from E. coli extracts. Upper row, kinase assays against Histone H1 with plant proteins purified from bacteria. Complexes binding to a Ni-NTA column (His-tagged proteins) were purified from cells either expressing HisGST-tagged CYCD3;1 together with Cak1, or HisGST-CYCD3;1 together with StrepIII-tagged CDKA;1 or cells expressing all three components, i.e. CYCD3;1, CDKA;1 and Cak1. Only complexes from cells co-expressing all three proteins showed high levels of kinase activity demonstrating the requirement of T-loop phosphorylation of CDK-cyclin complexes. Note that the bacteria system has a very little background kinase activity. Lower row, the same SDS-PAGE gel stained with coomassie brilliant blue (CBB) reveals equal loading of Histone H1. Abbreviations: CBB Coomassie brilliant blue. p-Histone H1 for radio-labeled Histone H1 resulting from kinase assays with radio-labeled ATP.