Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AHP2, a signal transmitter protein from Arabidopsis thaliana
| Autoři | |
|---|---|
| Rok publikování | 2013 |
| Druh | Článek v odborném periodiku |
| Časopis / Zdroj | Acta Crystalographica Section F |
| Fakulta / Pracoviště MU | |
| Citace | |
| www | http://journals.iucr.org/f/issues/2013/02/00/gj5115/stdsup.html |
| Doi | http://dx.doi.org/10.1107/S174430911205186X |
| Obor | Biochemie |
| Klíčová slova | multistep phosphorelay; AHP2; thermal shift assay; crystallization; vapor diffusion method; X-ray diffraction |
| Přiložené soubory | |
| Popis | 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. |
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