Purification and cDNA cloning of cytokinin-specific binding protein from mung bean (Vigna radiata)
Purification and cDNA cloning of cytokinin-specific binding protein from mung bean (Vigna radiata)
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DOI：
10.1046/j.1432-1327.1998.2580794.x
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发表时间：
1998-12-01
期刊：
影响因子：
--
通讯作者：
Hashimoto, Y
中科院分区：
文献类型：
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作者：
Fujimoto, Y;Nagata, R;Hashimoto, Y
合成的尿素衍生物如N-苯基-N′-（4-吡啶基）尿素（4PU）和N-（2-氯-4-吡啶基）-N′-苯脲（4PU30）具有较强的细胞分裂素活性。使用tritriated 4PU30作为探针，我们先前在黄化绿豆幼苗的可溶性部分中建立了高亲和力（K-a对4PU30 = 4X10(10) M-1）的细胞分裂素特异性结合蛋白（CSBP） [Nagata， R., Kawachi， E., Hashimoto， Y. & Shudo， K.] (1993) Biochem。Biophys。参考文献。1999,543 -549]。在本报告中，我们使用4PU- sepharose 4B（一种与4PU配体的亲和凝胶）纯化了CSBP。我们测定了CSBP的部分氨基酸序列，并通过RT - PCR分离了其cDNA。cDNA编码的蛋白分子量约为17 kDa。数据库同源性研究表明，CSBP是花粉过敏原/致病相关蛋白家族的新成员。重组CSBP在大肠杆菌中表达，并被证实与细胞分裂素特异性结合。
Synthetic urea derivatives such as N-phenyl-N'-(4-pyridyl)urea (4PU) and N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30) have strong cytokinin activities. Using tritiated 4PU30 as a probe, we previously established the presence of a cytokinin-specific binding protein (CSBP) of high affinity (K-a for 4PU30 = 4X10(10) M-1) in the soluble fraction of etiolated mung bean seedlings [Nagata, R., Kawachi, E., Hashimoto, Y. & Shudo, K. (1993) Biochem. Biophys. Res. Commun. 191, 543-549]. In this report, we purified CSBP by the use of 4PU-Sepharose 4B, an affinity gel liganded with 4PU. We determined partial amino acid sequences of CSBP and isolated its cDNA by reverse-transcription (RT PCR. The cDNA encoded a protein with a calculated molecular mass of 17 kDa, A data base homology search revealed that CSBP is a novel member of a major pollen allergen/pathogenesis-related protein family. Recombinant CSBP was expressed in Escherichia coli and was confirmed to bind specifically to cytokinins.