Dipetalogastin, a potent thrombin inhibitor from the blood-sucking insect Dipetalogaster maximus -: cDNA cloning, expression and characterization
Dipetalogastin, a potent thrombin inhibitor from the blood-sucking insect Dipetalogaster maximus -: cDNA cloning, expression and characterization
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DOI：
10.1046/j.1432-1327.1999.00895.x
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发表时间：
1999-12-01
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影响因子：
--
通讯作者：
Nowak, G
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文献类型：
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作者：
Mende, K;Petoukhova, O;Nowak, G
从吸血昆虫 Dipetalogaster maximus 的胃文库中分离出了编码凝血酶抑制剂二倍胃素的 cDNA。克隆的抑制剂 cDNA 的开放阅读框编码 344 个氨基酸残基的蛋白质。序列分析揭示了三个重复同源主要区域的存在，表明该抑制剂由三个结构域组成：每个结构域均显示出具有内部序列同源性的双头结构，如来自吸血昆虫Rhodnius probixus的凝血酶抑制剂Rhodniin。推导的氨基酸序列的肽序列比较显示结构域I和II分别与来自D.maximus胃内容物的天然抑制剂二古洛加斯汀和罗丹宁具有高度同源性。与 Kazal 型抑制剂显着的序列相似性，如保守序列 CGXDXXTYXNXC 和几个半胱氨酸残基，表明来自大蠹的凝血酶抑制剂是属于 Kazal 型家族的另一种吸血昆虫（除了杜鹃素）。在大肠杆菌中表达了对应于二倍加斯汀 cDNA 结构域 II 的具有生物活性的重组蛋白。 The isolated recombinant dipetalogastin with a molecular mass of,12.91 kDa has proved to be a specific thrombin inhibitor similar to its natural counterpart as well as rhodniin and hirudin.重组二倍加斯汀的 K-i 值测定为 49.3 +/- 22.28 fM。
A cDNA coding for the thrombin inhibitor dipetalogastin has been isolated from a stomach library of Dipetalogaster maximus, a blood-sucking insect. The open reading frame of the cloned inhibitor cDNA codes for a Protein of 344 amino-acid residues. Sequence analysis reveals the existence of three repeated homologous main regions, indicating that the inhibitor consists of three domains: Each domain shows a double-headed structure with an internal sequence homology like rhodniin, the thrombin inhibitor from the blood-sucking insect Rhodnius probixus. Peptide sequence comparisons of the deduced amino-acid sequence exhibit a high homology of the domains I and Il to the natural inhibitor dipetalogastin from the stomach content of D. maximus and to rhodniin, respectively. Significant sequence similarities to Kazal-type inhibitors, like the conserved sequence CGXDXXTYXNXC and several cysteine residues, indicate that the thrombin inhibitor from D. maximus is a further blood-sucking insect which belongs to the Kazal-type:family (besides rhodniin). A biologically active recombinant protein corresponding to domain II of the dipetalogastin cDNA was expressed in Escherichia coli. The isolated recombinant dipetalogastin with a molecular mass of,12.91 kDa has proved to be a specific thrombin inhibitor similar to its natural counterpart as well as rhodniin and hirudin. The K-i value of the recombinant dipetalogastin was determined to be 49.3 +/- 22.28 fM.