PMID:10497067
| Citation |
Li, Z and Crooke, E (1999) Functional analysis of affinity-purified polyhistidine-tagged DnaA protein. Protein Expr. Purif. 17:41-8 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1006/prep.1999.1094 |
| Keywords |
Amino Acid Sequence; Bacterial Proteins/genetics; Bacterial Proteins/isolation & purification; Bacterial Proteins/metabolism; Chromatography, Affinity; DNA Replication; DNA-Binding Proteins/genetics; DNA-Binding Proteins/isolation & purification; DNA-Binding Proteins/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Gene Expression; Genes, Bacterial; Histidine; Peptides/chemistry; Peptides/genetics; Plasmids/genetics; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism |
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