Herbaspirillum seropedicae is an endophytic, diazotroph, gram-negative, vibrioid bacterium of the β-Proteobacteria subdivision. This bacterium is found associated with grasses of economic interest such as maize, rice and sugarcane. The ntrY and ntrX genes constitute a two-component regulatory system, where NtrY is a sensory kinase protein and NtrX is a transcriptional activator. The NtrYX proteins are probably involved in the regulation of nitrate metabolism and/or transport in H. seropedicae. The N-terminal region of NtrX has a REC domain that receives the signal from the sensor partner by phosphorylation and the C-terminus has a DNA binding helix-turn-helix (HTH) motif. In this study the NtrX protein of H. seropedicae was over-expressed and purified. The ntrX gene was amplified and cloned into the expression vector pET28a, producing the plasmid pJHO2. This plasmid was transformed into an E. coli strain BL21 (lDE) and the NtrX expression was induced by IPTG (0.5 mmol/L) or lactose (0.5%) for 3h at 30 ⁰C. The cells were harvested by centrifugation and the cell pellet was resuspended in sonication buffer. After lysis the cell extract was centrifuged and the supernatant and precipitate fractions were analyzed by 12% SDS-PAGE. The His-tagged NtrX migrated on SDS-PAGE with an apparent molecular mass of 28.7kDa. Induction with IPTG resulted in higher yield of NtrX-His but most of the expressed protein was insoluble. When lactose was used as the inducer 58% of the expressed protein was soluble. This soluble fraction was purified by affinity chromatography on a HiTrap^TMChelating HP column. The His-tag protein was eluted with 240 - 280 mmol/L of imidazole.
Supported by CNPq/MCT/Pronex, Fundação Araucária and Paraná Tecnologia