Degtjarik Oksana, Dopitova Radka, Puehringer Sandra, Nejedla Eliska, Kuty Michal, Weiss Manfred S, Hejatko Jan, Janda Lubomir, Kuta Smatanova Ivana
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.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 Feb 1;69(Pt 2):158-61. doi: 10.1107/S174430911205186X. Epub 2013 Jan 31.
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.
拟南芥中含组氨酸的磷转移蛋白（AHP1 - 5）在一种称为多步磷酸化信号转导（MSP）的信号系统中，作为传感组氨酸激酶和响应调节因子之间的中间体。AHP蛋白介导并可能整合各种基于MSP的信号通路（例如细胞分裂素或渗透压感应）。然而，关于AHP蛋白的结构信息及其在MSP信号传导中的重要性仍然缺乏。为了更深入地了解AHP介导的信号转导的结构基础，我们确定了AHP2的三维结构。将AHP2编码序列克隆到pRSET B表达载体中，使得能够产生与N端His标签融合的AHP2。AHP2在大肠杆菌菌株BL21（DE3）pLysS中以可溶形式表达，然后在还原条件下使用金属螯合亲和色谱和阴离子交换色谱纯化至同质。在针对热稳定性优化的缓冲液中成功结晶，得到了衍射分辨率为2.5 Å的晶体。
