Expression, purification, and DNA-binding activity of the solubilized NtrC protein of Herbaspirillum seropedicae

• Published in: Protein expression and purification 2003-07, Vol.30 (1), p.117-123
• Main Authors: Twerdochlib, Adriana L, Chubatsu, Leda S, Souza, Emanuel M, Pedrosa, Fábio O, Steffens, M.Berenice R, Yates, M.Geoffrey, Rigo, Liu U
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; DNA - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; Electrophoretic Mobility Shift Assay; Gene Expression; Herbaspirillum; Histidine; PII Nitrogen Regulatory Proteins; Protein Structure, Tertiary; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Solubility; Trans-Activators; Transcription Factors - chemistry; Transcription Factors - isolation & purification; Transcription Factors - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/S1046-5928(03)00074-3

NtrC is a bacterial enhancer-binding protein (EBP) that activates transcription by the σ 54 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.