Figure 1

Interactions and oligomeric states of co-expressed fragments of Mcm4, 6 and 7. (A) Schematic of the polycistronic co-expression strategy that involves two compatible vectors. ORF1 and ORF2 were linked by a ribosome binding site (RBS) with a spacer. ORF3 was cloned in pXA-BN vector. Two plasmids were co-transformed into E. coli., followed by dual screening of ampicillin (50 μg/ml) and chloramphenicol (17 μg/ml). (B) Interactions of co-expressed and copurified fragments of Mcm4, 6 and 7, as identified in the two components co-expression (left side) or three components co-expression (right side) experiments. E. coli. lysates co-expressing various fragments with or without tags were passed through either glutathione or Ni-NTA resins, then the resins were washed as described under “Materials and Methods”. GST tags were cleaved by PreScission protease on the resin to release the MCM proteins. His tagged proteins were eluted by imidazole. All elutions were analyzed by SDS-PAGE. Asterisk denotes the co-lysis (instead of co-expression) of the indicated near-full-length fragments.