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Studies on production of a thrombin-like snake venom enzyme using a recombinant DNA

利用重组DNA生产类凝血酶蛇毒酶的研究

基本信息

批准号：
60880020
负责人：
YAMASHINA Ikuo
金额：
$ 10.11万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1987
项目状态：
已结题

项目摘要

Batroxobin is a thrombin-like enzyme, isolated from a snake Bothrops atrox, moojeni venom. Umlike thrombin which releases fibrinopeptides A and B from fibrinogen, this enzyme teleases only fibrinopepride A. Because of its defibrinogenerating effect, this enzyme is currently used clinically for the treatment of thrombotic diseases. We isolated cDNA from the venom gland cDNA library. Determination of the nucleotide sequence of the cDNA allowed elucidation of the complete amino acid sequence of batroxobin, the first time for a thrombin-like snake venom enzyme. The amino acid sequence of batroxobin exhibited significant homology with those of eukaryotic serine proteases, indeicating that batroxobin is a member of the serine protease family. We habe investigated betroxobin based on gene construction to chatacterize more this enzyme. Using the batroxobin cDNA we have isolated three overlapping DNA segments containing the entire batroxobin gene. Sequence analysis revealed that the batroxobin gene spans 8 kbp and contains five exons. The mature batroxobin is encoded by four separate exons, 2 to 5. The catalytic residues of baroxobin, His-41, Asp-86 and Ser-178, are encoded by seperate exons, 2, 3 and 5, respectively.The exon/intron organixzation of the batroxobin gene is different from that of the prothrombin gene, bur evry similar to those of the trypsin and kallikrein genes. The snake venom gland is assumed to originate from the submaxillary gland. Therefore, batroxobin is expected to be a member of the glandular kallikrein family.Expression of batroxobin in E. coli cells plasmide of which included the batroxobin cDNA linked to a fragment of the bactor XIII cDNA has been followed. Several mg of the polypeptide reactive with the batroxobin antibody was in fact prouduced in one liter culture, of which about 20 % could be conberted to an enzymatically active form by in vitro refolding of the disulfide bounds.

Batroxobin是一种类似凝血酶的酶，从一种叫做Bothrops atrox的蛇毒中分离出来。与从纤维蛋白原释放纤维蛋白肽A和B的凝血酶不同，这种酶只释放纤维蛋白肽A。由于它的去纤维蛋白生成作用，这种酶目前在临床上用于治疗血栓性疾病。我们从毒腺cDNA文库中分离cDNA。cDNA的核苷酸序列测定使batroxobin的完整氨基酸序列得以阐明，这是第一次对类似凝血酶的蛇毒酶进行分析。batroxobin的氨基酸序列与真核丝氨酸蛋白酶具有显著的同源性，表明batroxobin是丝氨酸蛋白酶家族的成员。我们已经研究了基于基因结构的贝曲酶，以更多地表征这种酶。利用batroxobin cDNA，我们分离出三个重叠的DNA片段，包含整个batroxobin基因。序列分析显示，batroxobin基因全长8 kbp，包含5个外显子。成熟的batroxbin由4个独立的外显子2到5编码。baroxobin的催化残基His-41、Asp-86和Ser-178分别由独立的外显子2、3和5编码。batroxobin基因的外显子/内含子组织与凝血酶原基因不同，但与胰蛋白酶和钾化酶基因非常相似。蛇毒腺被认为起源于颌下腺。因此，batroxobin有望成为腺激肽激酶家族的一员。在大肠杆菌细胞质粒中，其中含有与细菌XIII cDNA片段连接的batroxobin cDNA的表达已被跟踪。事实上，在一升培养物中产生了几毫克与细胞氧化酶抗体反应的多肽，其中约20%可以通过体外二硫键的重折叠转化为酶活性形式。