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 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.