Histidine-containing phosphotransfer proteins from Arabidopsis thaliana (AHP1-5) act as intermediates between sensor histidine kinases and response regulators in a signalling system called multi-step phosphorelay (MSP). AHP proteins mediate and potentially integrate various MSP-based signalling pathways (e.g. cytokinin or osmosensing). However, structural information about AHP proteins and their importance in MSP signalling is still lacking. To obtain a deeper insight into the structural basis of AHP-mediated signal transduction, the three-dimensional structure of AHP2 was determined. The AHP2 coding sequence was cloned into pRSET B expression vector, enabling production of AHP2 fused to an N-terminal His tag. AHP2 was expressed in soluble form in Escherichia coli strain BL21 (DE3) pLysS and then purified to homogeneity using metal chelate affinity chromatography and anion-exchange chromatography under reducing conditions. Successful crystallization in a buffer which was optimized for thermal stability yielded crystals that diffracted to 2.5 Å resolution.
- MeSH
- Arabidopsis metabolism MeSH
- X-Ray Diffraction MeSH
- Electrophoresis, Polyacrylamide Gel MeSH
- Phosphotransferases chemistry isolation & purification MeSH
- Crystallization MeSH
- Arabidopsis Proteins chemistry isolation & purification MeSH
- Signal Transduction * MeSH
- Transition Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
