Infestin, a thrombin inhibitor presents in Triatoma infestans midgut, a Chagas' disease vector:: gene cloning, expression and characterization of the inhibitor
Infestin, a thrombin inhibitor presents in Triatoma infestans midgut, a Chagas' disease vector:: gene cloning, expression and characterization of the inhibitor
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DOI：
10.1016/s0965-1748(02)00035-8
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发表时间：
2002-09-01
影响因子：
3.8
通讯作者：
Tanaka, AS
中科院分区：
文献类型：
--
作者：
Campos, ITN;Amino, R;Tanaka, AS
这项工作描述了 Infestin（一种来自 Triatoma infestans 中肠的凝血酶抑制剂）的纯化、基因克隆和表达。 Infestin 位于中肠，通过阴离子交换和亲和色谱进行纯化。确定了N-末端序列和胰蛋白酶肽的序列。使用RT-PCR、总RNA和infestin cDNA信息，克隆了编码多种非经典Kazal型丝氨酸蛋白酶抑制剂的DNA片段。分离的天然 infestin 具有两个非经典 Kazal 型结构域，显示出 13 kDa 的表观分子量，而其基因编码的蛋白质具有四个非经典 Kazal 型结构域，对应于 22 kDa 的表观分子量。使用载体pVT102U/α构建两种重组infestin，r-infestin 1-2和r-infestin 1-4，并在酿酒酵母中表达。 Native 和 r-infestin 1-2 对凝血酶和胰蛋白酶表现出非常相似的抑制活性，凝血酶的解离常数分别为 43.5 和 25 pM，胰蛋白酶的解离常数分别为 2.0 和 3.1 nM。 r-infestin 1-2 没有抑制凝血级联的其他丝氨酸蛋白酶。令人惊讶的是，r-infestin 1-4 不仅抑制凝血酶和胰蛋白酶（Ki 分别为 0.8 和 5.2 nM），而且还抑制因子 XIIa、因子 Xa 和纤溶酶（K-i 分别为 78 pM、59.2 和 1.1 nM）。 (C) 2002 Elsevier Science Ltd. 保留所有权利。
This work describes the purification, gene cloning and expression of infestin, a thrombin inhibitor from midguts of Triatoma infestans. Infestin is located in the midgut and its purification was performed by anion-exchange and affinity chromatographies. The N-terminal sequence and the sequence of tryptic peptides were determined. Using RT-PCR, total RNA and infestin cDNA information, a DNA fragment was cloned which encodes a multi non-classical Kazal-type serine protease inhibitor. Isolated native infestin has two non-classical Kazal-type domains and shows an apparent molecular mass of 13 kDa, while its gene codes for a protein with four non-classical Kazal-type domains corresponding to an apparent molecular mass of 22 kDa. Two recombinant infestins, r-infestin 1-2 and r-infestin 1-4, were constructed using the vector pVT102U/alpha and expressed in S. cerevisiae. Native and r-infestin 1-2 showed very similar inhibitory activities towards thrombin and trypsin with dissociation constants of 43.5 and 25 pM for thrombin and 2.0 and 3.1 nM for trypsin, respectively. No other serine protease of the blood coagulation cascade was inhibited by the r-infestin 1-2. Surprisingly, r-infestin 1-4 inhibited not only thrombin and trypsin (Ki of 0.8 and 5.2 nM, respectively), but also factor XIIa, factor Xa and plasmin (K-i of 78 pM, 59.2 and 1.1 nM, respectively). (C) 2002 Elsevier Science Ltd. All rights reserved.