title: Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AHP2, a signal transmitter protein from Arabidopsis thaliana.
abstract: 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.
authors: South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses and School of Complex Systems, University of South Bohemia, Zamek 136, 37333 Nove Hrady, Czech Republic.
authors: Oksana
authors: O
authors: Degtjarik
authors: Radka
authors: R
authors: Dopitova
authors: Sandra
authors: S
authors: Puehringer
authors: Eliska
authors: E
authors: Nejedla
authors: Michal
authors: M
authors: Kuty
authors: Manfred S
authors: MS
authors: Weiss
authors: Jan
authors: J
authors: Hejatko
authors: Lubomir
authors: L
authors: Janda
authors: Ivana
authors: I
authors: Kuta Smatanova
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D010770:Phosphotransferases / Q000737:chemistry* / Q000302:isolation & purification*; D015398:Signal Transduction; D044366:Transition Temperature; D014961:X-Ray Diffraction
chemical_list: C117967:AHP2 protein, Arabidopsis; D029681:Arabidopsis Proteins; D010770:Phosphotransferases
keywords: AHP2; Arabidopsis thaliana; histidine phosphotransfer proteins; multi-step phosphorelay
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