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

Degtjarik Oksana; Dopitova Radka; Puehringer Sandra; Nejedla Eliska; Kuty Michal; Weiss Manfred S.; Hejatko Jan; Janda Lubomir; Smatanova Ivana Kuta

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

机译：拟南芥信号传递蛋白AHP2的克隆，表达，纯化，结晶和初步X射线衍射分析

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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 angstrom resolution.

机译：来自拟南芥的含组氨酸的磷酸转移蛋白（AHP1-5）在称为多步磷酸化（MSP）的信号系统中，充当传感器组氨酸激酶和响应调节剂之间的中间体。 AHP蛋白介导并可能整合各种基于MSP的信号传导途径（例如细胞分裂素或渗透压）。但是，仍然缺乏有关AHP蛋白质及其在MSP信号转导中的重要性的结构信息。为了更深入地了解AHP介导的信号转导的结构基础，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，从而能够生产与N末端His标签融合的AHP2。 AHP2以可溶形式在大肠杆菌BL21（DE3）pLysS中表达，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均一。在针对热稳定性进行了优化的缓冲液中成功结晶产生了衍射至2.5埃分辨率的晶体。

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《Acta crystallographica, Section F. Structural biology and crystallization communications》 |2013年第2期|共4页
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Degtjarik Oksana; Dopitova Radka; Puehringer Sandra; Nejedla Eliska; Kuty Michal; Weiss Manfred S.; Hejatko Jan; Janda Lubomir; Smatanova Ivana Kuta;
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1. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AHP2, a signal transmitter protein from Arabidopsis thaliana [J] . Degtjarik Oksana, Dopitova Radka, Puehringer Sandra, Acta crystallographica, Section F. Structural biology and crystallization communications . 2013,第2期

机译：拟南芥信号传递蛋白AHP2的克隆，表达，纯化，结晶和初步X射线衍射分析
2. Expression, purification, crystallization and preliminary X-ray diffraction analysis of Arabidopsis thaliana Deg8 [J] . Shan Xiaoyue, Sun Wei, Fan Haitian, Acta crystallographica, Section F. Structural biology and crystallization communications . 2013,第1期

机译：拟南芥Deg8的表达，纯化，结晶和初步X射线衍射分析
3. GPX3 from Arabidopsis thaliana: cloning, expression, purification, crystallization and preliminary X-ray analysis [J] . Li Kun, Yang Qingzhan, Wang Wei, Acta crystallographica, Section F. Structural biology and crystallization communications . 2013,第11期

机译：拟南芥GPX3：克隆，表达，纯化，结晶和初步X射线分析
4. Comparative Differential Gene Expression Analysis of Nucleus-encoded Proteins for Rafflesia cantleyi against Arabidopsis thaliana [C] . Siuk-Mun Ng, Xin-Wei Lee, Kiew-Lian Wan, UKM FST Postgraduate Colloquium . 2015

机译：rafflesia contleyi对拟南芥的细胞核编码蛋白的比较差异基因表达分析
5. Stringent starvation protein A: Expression, purification, characterization, and preliminary x-ray diffraction studies [D] . Andrykovitch, Michelle 2002

机译：严格的饥饿蛋白A：表达，纯化，表征和初步X射线衍射研究
6. Cloning expression purification crystallization and preliminary X-ray diffraction analysis of AHP2 a signal transmitter protein from Arabidopsis thaliana [O] . Oksana Degtjarik, Radka Dopitova, Sandra Puehringer, 2013

机译：克隆表达纯化结晶和aHp2来自拟南芥的信号发射机蛋白的初步X射线衍射分析
7. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the apo form of InsP 5 2-K from Arabidopsis thaliana [O] . Baños-Sanz, J.I., Sanz-Aparicio, J., J. Brearley, C.A., 2013

机译：拟南芥InsP 5 2-K载脂蛋白形式的表达，纯化，结晶和初步X射线衍射分析

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

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