title: Expression, purification, and DNA-binding activity of the solubilized NtrC protein of Herbaspirillum seropedicae.
abstract: NtrC is a bacterial enhancer-binding protein (EBP) that activates transcription by the sigma54 RNA polymerase holoenzyme. NtrC has a three domain structure typical of EBP family. In Herbaspirillum seropedicae, an endophytic diazotroph, NtrC regulates several operons involved in nitrogen assimilation, including glnAntrBC. In order to over-express and purify the NtrC protein, DNA fragments containing the complete structural gene for the whole protein, and for the N-terminal+Central and Central+C-terminal domains were cloned into expression vectors. The NtrC and NtrC(N-terminal+Central) proteins were over-expressed as His-tag fusion proteins upon IPTG addition, solubilized using N-lauryl-sarcosyl and purified by metal affinity chromatography. The over-expressed His-tag-NtrC(Central+C-terminal) fusion protein was partially soluble and was also purified by affinity chromatography. DNA band-shift assays showed that the NtrC protein and the Central+C-terminal domains bound specifically to the H. seropedicae glnA promoter region. The C-terminal domain is presumably necessary for DNA-protein interaction and DNA-binding does not require a phosphorylated protein.
authors.affiliation: Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, Caixa Postal 19046, Curitiba, PR 81531-990, Brazil.
authors.forename: Adriana L
authors.initials: AL
authors.lastname: Twerdochlib
authors.forename: Leda S
authors.initials: LS
authors.lastname: Chubatsu
authors.forename: Emanuel M
authors.initials: EM
authors.lastname: Souza
authors.forename: Fábio O
authors.initials: FO
authors.lastname: Pedrosa
authors.forename: M Berenice R
authors.initials: MB
authors.lastname: Steffens
authors.forename: M Geoffrey
authors.initials: MG
authors.lastname: Yates
authors.forename: Liu U
authors.initials: LU
authors.lastname: Rigo
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D042803:Herbaspirillum; D006639:Histidine; D051922:PII Nitrogen Regulatory Proteins; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D015534:Trans-Activators; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D051922:PII Nitrogen Regulatory Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D006639:Histidine; D004247:DNA