Publication details

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AHP2, a signal transmitter protein from Arabidopsis thaliana

Authors

DEGTJARIK Oksana DOPITOVÁ Radka PUEHRINGER Sandra NEJEDLÁ Eliška KUTÝ Michal WEISS Manfred HEJÁTKO Jan JANDA Lubomír KUTÁ-SMATANOVÁ Ivana

Year of publication 2013
Type Article in Periodical
Magazine / Source Acta Crystalographica Section F
MU Faculty or unit

Central European Institute of Technology

Citation
Web http://journals.iucr.org/f/issues/2013/02/00/gj5115/stdsup.html
Doi http://dx.doi.org/10.1107/S174430911205186X
Field Biochemistry
Keywords multistep phosphorelay; AHP2; thermal shift assay; crystallization; vapor diffusion method; X-ray diffraction
Attached files
Description 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 A resolution.
Related projects:

You are running an old browser version. We recommend updating your browser to its latest version.

More info