Expression, purification, and functional analysis of the C-terminal domain of Herbaspirillum seropedicae NifA protein

• Published in: Protein expression and purification 2003-02, Vol.27 (2), p.313-318
• Main Authors: Monteiro, Rose A, Souza, Emanuel M, Geoffrey Yates, M, Steffens, M.Berenice R, Pedrosa, Fábio O, Chubatsu, Leda S
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Bacterial Proteins - physiology; Betaproteobacteria - metabolism; Chromatography; Cloning, Molecular; DNA - metabolism; Escherichia coli - metabolism; Herbaspirillum seropedicae; Klebsiella pneumoniae - genetics; NifA protein; Nitrogen fixation; Plasmids - metabolism; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; Recombinant Fusion Proteins - metabolism; Transcription Factors - chemistry; Transcription Factors - isolation & purification; Transcription Factors - physiology; Transcription, Genetic; Transcriptional Activation; Transcriptional activator
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/S1046-5928(02)00635-6

The Herbaspirillum seropedicae NifA protein is responsible for nif gene expression. The C-terminal domain of the H. seropedicae NifA protein, fused to a His-Tag sequence (His-Tag-C-terminal), was over-expressed and purified by metal-affinity chromatography to yield a highly purified and active protein. Band-shift assays showed that the NifA His-Tag-C-terminal bound specifically to the H. seropedicae nifB promoter region in vitro. In vivo analysis showed that this protein inhibited the Central + C-terminal domains of NifA protein from activating the nifH promoter of K. pneumoniae in Escherichia coli, indicating that the protein must be bound to the NifA-binding site (UAS site) at the nifH promoter region to activate transcription.