Expression, purification, and DNA-binding activity of the solubilized NtrC protein of Herbaspirillum seropedicae.
Protein Expr Purif
; 30(1): 117-23, 2003 Jul.
Article
em En
| MEDLINE
| ID: mdl-12821329
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
/
Transativadores
/
Herbaspirillum
/
Proteínas de Ligação a DNA
Idioma:
En
Revista:
Protein Expr Purif
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2003
Tipo de documento:
Article
País de afiliação:
Brasil
País de publicação:
Estados Unidos