Functional analysis of affinity-purified polyhistidine-tagged DnaA protein

DnaA protein initiates DNA replication at the Escherichia coil chromosomal origin. We describe a system for efficient production and purification of r eplicatively active DnaA protein. The dnaA gene was cloned in-frame with a sequence encoding a polyhistidine tag and expressed from a T7 promoter regu lated by the lac operator. DnaA with the amino terminal polyhistidine tag w as isolated using immobilized metal-ion affinity chromatography. Immunoblot analysis indicated that the tagged protein was intact and migrated with th e expected molecular weight. The yield of purified protein was greater than 10 mg per liter of cell culture. The polyhistidine-tagged DnaA protein was comparable to nontagged DnaA protein for initiating in vitro DNA replicati on, binding to oriC DNA, binding of allosteric effector adenine nucleotides , and interaction with membrane acidic phospholipids. This system for rapid and high-yield generation of replication-active DnaA protein should facili tate structure-function studies and mutagenic analyses of this initiator pr otein. (C) 1999 Academic Press.