Crystallization of ExoR - His6, A Regulatory Signal Controlling Symbiotic Nitrogen Fixation
Year: 2025
Type: conference-abstract
Abstract: ExoR is a signaling protein that controls nitrogen fixation, the conversion of N2 gas into ammonia fertilizer. The ExoR protein in the soil bacterium Sinorhizobium meliloti acts as a novel inhibitor of the two-component regulatory system (ExoS/ChvI), blocking the production of the signaling molecule succinoglycan, which is needed for establishing a nitrogen-fixing symbiosis with the legume. Previous attempts to determine the crystal structure of ExoR have been unsuccessful, due to the tendency of the protein to precipitate at protein concentrations above 3 mg/mL. The goal of the current project is to develop a purification strategy to produce crystallization-quality ExoR protein. Histidine-tagged ExoR (ExoR-His6) was produced in Escherichia coli and purified in two steps using nickel affinity (NiNTA) chromatography and S200 gel filtration chromatography. After purification, Bradford assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) showed that the first NiNTA elution sample (100 mM imidazole) contained protein at a concentration of 1.35 mg/mL and a purity of ∼46%. After the S200 gel filtration column, the protein concentration was 0.21 mg/mL, and the purity increased to ∼90%. Altering the collected volumes to modify elution conditions resulted in a 2-fold increase in ExoR concentration. Crystallization using a 1 M ammonium sulfate buffer successfully produced a 100-micron crystal, confirming that a higher protein concentration was benficial for larger, diffraction-quality crystals. Experiments are in progress to increase the purity of the protein to greater than 98%, in order to determine the three-dimensional structure of the protein.
Source: Structural Dynamics
DOI: 10.1063/4.0000837
Authors: Katherine Lee, Kenneth Childers, Madeline Rasche
Institution: California State University, Fullerton
Language: English
Subfield: Materials Chemistry
Field: Materials Science
Domain: Physical Sciences
Open Access status: gold
Estimated APC paid: $2,200
OpenAlex ID: W7101385474
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