Expression, purification, and DNA-binding activity of the solubilized NtrC protein of Herbaspirillum seropedicae.
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.
Twerdochlib
Adriana L
AL
Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, Caixa Postal 19046, Curitiba, PR 81531-990, Brazil.
Chubatsu
Leda S
LS
Souza
Emanuel M
EM
Pedrosa
Fábio O
FO
Steffens
M Berenice R
MB
Yates
M Geoffrey
MG
Rigo
Liu U
LU
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
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