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 lambda O 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. (C) 2016 Elsevier Inc. All rights reserved.