The cloning and overexpression of a cruciform binding protein from Ustilago maydis
The cloning and overexpression of a cruciform binding protein from Ustilago maydis
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DOI：
10.1016/s0167-4781(96)00227-8
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发表时间：
1997-06-26
期刊：
影响因子：
--
通讯作者：
Kmiec, EB
中科院分区：
文献类型：
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作者：
Dutta, S;Gerhold, DL;Kmiec, EB
编码玉米黑粉菌十字形DNA结合蛋白的结构基因HMP1已被克隆。使用根据从纯化蛋白质获得的氨基酸序列设计的引物，通过聚合酶链式反应程序实现基因分离。 DNA序列测定表明该基因编码含有98个氨基酸的蛋白质，计算分子量为10151。cDNA和基因组序列的比较表明该基因的5'编码区存在单个内含子。该基因作为与六组氨酸前导序列的翻译融合体进行过表达，从而能够在固定金属基质上亲和纯化蛋白质。过表达后分离的蛋白质表现出十字形结合活性，符合早期纯化的天然蛋白质结果。序列分析表明不存在 HMG 盒，并且与其他已知的十字形结合蛋白几乎没有同源性。 HMP1 可能代表了一类能够识别此类二级结构的新型蛋白质。 (C) 1997 Elsevier Science B.V.
The structural gene HMP1 encoding a cruciform DNA binding protein from Ustilago maydis has been cloned. Gene isolation was enabled by a polymerase chain reaction procedure using primers designed from amino acid sequence obtained from the purified protein. DNA sequence determination has revealed that the gene encodes a protein containing 98 amino acids with a calculated molecular weight of 10151. Comparison of the cDNA and genomic sequences indicated the presence of a single intron in the 5' coding region of the gene. The gene was over-expressed as a translational fusion with a hexahistidine leader sequence enabling affinity purification of the protein on an immobilized metal matrix. Protein isolated after over-expression exhibited cruciform binding activity, conforming earlier purified native protein results. Sequence analysis indicated that no HMG box was present and very little homology to other known cruciform binding proteins was found. It is plausible that HMP1 represents a novel class of proteins that recognize such secondary structures. (C) 1997 Elsevier Science B.V.