Figure 1
From: Robust reconstitution of active cell-cycle control complexes from co-expressed proteins in bacteria
Scheme of the here-developed kinase assay procedure based on a Duet-vector system. (a) (1) CDK, e.g. CDKA;1, and cyclin, e.g. CYCD3;1, and CAK, e.g. Cak1 or CDKF;1, are co-expressed in E. coli and can be maintained due to the different replicons and the different resistances they confer, see also (b) and (c). (2) CDK-cyclin complexes, e.g. CDKA;1-CYCD3;1, are pulled-down through the His-tag on the cyclin partner. (3) Finally, kinase reactions are performed with the isolated complexes and complexes are in parallel subjected to western blotting with a Strep-Tactin-HRP detection system to determine the level of the CDK partner. (b) Generic Duet Vector with CloDF13 ori containing Cak1 or CDKF;1 (above) and Duet vector with CDK (CDKA;1/CDKB1;1/CDKB2;2) (below). (c) Generic Gateway-compatible Expression Vector with ColE1 ori (above) and vector with cyclin (CYCD3;1/CYCB1;2/SDS) (below).