Although recombinant protein expression in E. coli has become all but routine, high level protein expression or over-expression is not always consistent and repeatable for every protein. Our research at Zymo Research Corp. has shown that high level protein expression can be achieved consistently when two processes – cell expansion and protein expression – are completely separated. The Dual Media Set, different from commonly used protein expression procedures using Luria Broth (LB) or other specially prepared medium, contains two media, EXPANSION BROTH (EB) and OVEREXPRESSION BROTH (OB). For cell expansion, E. coli cells are grown in EB, while the production of the recombinant protein is almost completely repressed. For high level protein expression, the expanded cell culture is simply added into OB media. By using the Dual Media Set system, protein overexpression can be reliably controlled for many proteins.