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

巴曲酶是一种凝血酶样酶，从蛇毒中分离出来。Um型凝血酶从纤维蛋白原释放纤维蛋白多肽A和B，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，该酶目前被用于临床治疗血栓性疾病。我们从毒腺cDNA文库中分离得到了该基因。对该基因核苷酸序列的测定有助于阐明巴曲酶的完整氨基酸序列，这是首次发现类似凝血酶的蛇毒酶。巴曲酶的氨基酸序列与真核丝氨酸蛋白酶的氨基酸序列有很大的同源性，表明巴曲酶是丝氨酸蛋白酶家族的成员。为了进一步研究该酶的特性，我们在基因构建的基础上对其进行了研究。利用巴曲酶基因，我们分离到三个包含巴曲酶基因全长的重叠DNA片段。序列分析表明，巴曲酶基因全长8kbp，含有5个外显子。成熟的巴曲酶由4个独立的外显子2~5编码。巴曲酶的催化残基His-41、Asp-86和Ser-178分别由独立的外显子2、3和5编码。巴曲酶基因的外显子/内含子有机化不同于凝血酶原基因，但与胰酶和激肽释放酶基因相似。蛇毒腺被认为起源于颌下腺。因此，巴曲酶被认为是腺激肽释放酶家族的成员。巴曲酶在大肠杆菌细胞中的表达已被跟踪，其中包括与杆菌XIII基因片段相连的巴曲酶基因。事实上，在一升培养中可产生几毫克与巴曲酶抗体反应的多肽，其中约20%可通过体外二硫键重折叠而产生具有酶活性的形式。

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.