8S globulin of mungbean [Vigna radiata (L.) wilczek]:: Cloning and characterization of its cDNA isoforms, expression in Escherichia coli, purification, and crystallization of the major recombinant 8S isoform
8S globulin of mungbean [Vigna radiata (L.) wilczek]:: Cloning and characterization of its cDNA isoforms, expression in Escherichia coli, purification, and crystallization of the major recombinant 8S isoform
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DOI：
10.1021/jf0305938
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发表时间：
2004-05-05
影响因子：
6.1
通讯作者：
Tecson-Mendoza, EM
中科院分区：
文献类型：
--
作者：
Bernardo, AEN;Garcia, RN;Tecson-Mendoza, EM
绿豆主要8 S球蛋白cDNA的三种异构体，8 Salpha，8 Salpha '，和8 Sbeta，被分离、克隆和鉴定。8 Salpha、8 Salpha '和8 Sbeta的cDNA序列的开放阅读框分别为1362、1359或1362和1359 bp，分别编码454、453或454和453个氨基酸，相应的分子量分别为51973、51627或51758和51779。8 Salpha与8 Salpha '、8 Salpha与8 Sbeta、8 Salpha'与8 Sbeta的同源性分别为91-92%、87%，86-88%。使用signalP网站（Nielsen，H.; Engelbrecht，J.; Brunak，S.; von Heijne，G. 1997，10，1-6）。前肽被确定为IVHREN。在C端约90个氨基酸处观察到单一糖基化位点（N-X-S/T）。绿豆8 S异构体和其他7- 8 S蛋白之间的同源性在豆科植物中为45%至68%，在不同物种的作物中为29%至34%。在大肠杆菌中表达主要同种型8 Salpha，并通过连续的硫酸铵分级分离、疏水相互作用和Mono Q柱层析纯化。重组的8 Salpha，而不是天然形式，成功地结晶产生菱面体晶体。
Three isoforms of the cDNA of the major 8S globulin of mungbean, 8Salpha, 8Salpha', and 8Sbeta, were isolated, cloned, and characterized. The cDNA sequences of 8Salpha, 8Salpha', and 8Sbeta had open reading frames of 1362, 1359 or 1362, and 1359 bp, respectively, which code for 454, 453 or 454, and 453 amino acids corresponding to molecular weights of 51 973, 51 627 or 51 758, and 51 779, respectively. Homology in terms of cDNA and amino acid sequences was 91-92% between 8Salpha and 8Salpha', 87% between 8Salpha and 8Sbeta, and 86-88% between 8Salpha' and 8Sbeta. The signal peptide was found to be 1-25, 1-24 or 25, and 1-23 for 8Salpha, 8Salpha', and 8Sbeta, respectively, using the signalP website (Nielsen, H.; Engelbrecht, J.; Brunak, S.; von Heijne, G. Protein Eng. 1997, 10, 1-6). The propeptide was determined to be IVHREN. A single site for glycosylation (N-X-S/T) was observed about 90 amino acids from the C terminus. Homology between mungbean 8S isoforms and other 7-8S proteins ranged from 45 to 68% within members of the legume family and 29 to 34% for crops of different species. The major isoform 8Salpha was expressed in Escherichia coli and purified by successive ammonium sulfate fractionation, hydrophobic interaction, and Mono Q column chromatography. The recombinant 8Salpha, but not the native form, was successfully crystallized producing rhombohedral crystals.