Purified Stx and λ phage initiator O proteins bind specifically to two different origins of replication in vitro

•Improved method for overexpression of λ o gene and purification of its protein has been developed.•O proteins of Shiga toxin-converting phage P27 and 933W were purified for the first time.•All three proteins have been demonstrated to be active in binding to origin regions of bacteriophage λ and P27 genomes.

The O protein is a crucial factor initiating the DNA replication of lambdoid bacteriophage. Efficient DNA replication of Shiga toxin-converting phage is necessary for effective production of Shiga toxin - main virulence factor of STEC strains. We developed an improved protocol for overproduction, bacterial cell lysis and purification of λO protein. With use of this method we have also isolated O proteins of Stx-phage P27 and 933W that were never purified before. Purified proteins were tested for their DNA binding activity and revealed a sequence specific interactions.