PREPROABRIN - GENOMIC CLONING, CHARACTERIZATION AND THE EXPRESSION OF THE A-CHAIN IN ESCHERICHIA-COLI
PREPROABRIN - GENOMIC CLONING, CHARACTERIZATION AND THE EXPRESSION OF THE A-CHAIN IN ESCHERICHIA-COLI
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DOI：
10.1111/j.1432-1033.1991.tb16072.x
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发表时间：
1991-06-15
期刊：
影响因子：
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通讯作者：
PIATAK, M
中科院分区：
文献类型：
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作者：
WOOD, KA;LORD, JM;PIATAK, M
代表相思豆蛋白 C 的 N 端和 A 链内部区域的所有可能序列的合成寡核苷酸被用于通过对相思豆基因组 DNA 进行聚合酶链式反应来生成对相思豆蛋白相关序列特异的探针。对从 A. precatorius 叶组织中分离的基因组 DNA 构建的 lambda 噬菌体文库进行了筛选，并通过限制性内切酶分析对阳性杂交克隆进行了表征。通过 DNA 测序来表征独特克隆的编码区。一个克隆编码与相思豆蛋白 C 密切相关的前蛋白原，A 链序列之间有 83% 的相似性。基于与蓖麻毒素和蓖麻凝集素的相似性，前红豆蛋白原由 251 个氨基酸的 A 链组成，前面是包含 N 端信号肽的 34 个氨基酸，后面是 14 个氨基酸的接头和 263 个氨基酸的 B 链。前红豆蛋白原的成熟 A 链已在大肠杆菌中在细胞质中表达，并且可溶性重组蛋白的产生水平超过总细胞蛋白的 6%。重组A链已被纯化至均质，其在大鼠肝核糖体中脱嘌呤的能力已在体外得到证实。
Synthetic oligonucleotides representing all possible sequences of an N-terminal and an internal region of the A-chain of abrin C were used to generate a probe specific for abrin-related sequences using the polymerase chain reaction on Abrus precatorius genomic DNA. A lambda phage library constructed from genomic DNA isolated from leaf tissue of A. precatorius was screened and positive hybridising clones were characterised by restriction enzyme analysis. The coding regions of unique clones were characterised by DNA sequencing. One clone encodes a preproprotein closely related to abrin C with 83% similarity between the A-chain sequences. Based on similarity with the ricin toxins and Ricinus communis agglutinin, the preproabrin consists of an A-chain of 251 amino acids preceded by 34 amino acids containing an N-terminal signal peptide, followed by a 14-amino-acid linker and a B-chain of 263 amino acids. The mature A-chain of the preproabrin has been expressed cytoplasmically in Escherichia coli and the soluble recombinant protein was produced at levels exceeding 6% of total cell protein. The recombinant A-chain has been purified to homogeneity and its ability to depurinate 28S rRNA in rat liver ribosomes has been demonstrated in vitro.