Functional analysis of affinity-purified polyhistidine-tagged DnaA protein.
DnaA protein initiates DNA replication at the Escherichia coli chromosomal origin. We describe a system for efficient production and purification of replicatively active DnaA protein. The dnaA gene was cloned in-frame with a sequence encoding a polyhistidine tag and expressed from a T7 promoter regulated by the lac operator. DnaA with the amino terminal polyhistidine tag was isolated using immobilized metal-ion affinity chromatography. Immunoblot analysis indicated that the tagged protein was intact and migrated with the 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 replication, 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 facilitate structure-function studies and mutagenic analyses of this initiator protein.
Li
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Z
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Road Northwest, Washington, DC 20007, USA.
Crooke
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E
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D006639:Histidine; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D010455:Peptides; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine