The bacteriopbage λ O replication protein: isolation and characterization of the amplified initiator

Abstract

The bacteriophage λ 0 protein participates in the Initiation of λ DMA replication. The λ Q. gene was cloned into plasmid pKC30 ouch that its expression was controlled by the λ PL promoter. A λ prophage-coded thennosensitlve cI repressor was used to regulate transcription of the cloned Q. gene. Thermal inactivation of the λ cI repressor resulted in overproduction of the O protein until it constituted approximately 20% of the total cellular protein of Each-arlohla coll. A simple three-step purification protocol was developed that yields several milligrams of homogeneous O protein per gram of cell paste. The precise position of the Q. gene in the known λ DNA sequence was identified from the amino-terminal sequence of the isolated O protein. Purified O protein stimulated the replication of plasmid λdv DNA in vitro and specifically bound to duplex DNA fragments carrying the λ replication origin.