Purification, characterization and gene cloning of Da-36, a novel serine protease from Deinagkistrodon acutus venom

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• 作者：Ying Zheng^a ; ^b ; ¹ ; Feng-Ping Ye^b ; ^c ; ¹ ; Jie Wang^b ; Guo-Yang Liao^a ; Yun Zhang^c ; Quan-Shui Fan^b ; ^{fqs168@126.com} ; Wen-Hui Lee^c ; ^{leewh@mail.kiz.ac.cn}
• 关键词：Deinagkistrodon acutus ; Gene ; Serine protease ; Purification
• 刊名：Toxicon
• 出版年：2013
• 出版时间：1 June, 2013
• 年：2013
• 卷：67
• 期：Complete
• 页码：1-11
• 全文大小：3597 K

文摘

A serine protease termed Da-36 was isolated from crude venom of Deinagkistrodon acutus. The enzyme was a single chain protein with an apparent molecular weight of 36,000 on SDS-PAGE with an isoelectric point of 6.59. Da-36 could clot human plasma by cleaving the A¦Á, B¦Â and ¦Ã chains of fibrinogen and also exhibited arginine esterase activity. The proteolytic activity of Da-36 toward TAME was strongly inhibited by PMSF and moderately affected by benzamidine and aprotinin, indicating that it was a serine protease. Meanwhile, Da-36 showed stability with wide temperature (20-50?¡ãC) and pH value ranges (pH 6-10). Divalent metal ions of Ca²⁺, Mg²⁺, and Mn²⁺ had no effects but Zn²⁺ and Cu²⁺ inhibited the arginine esterase activity of Da-36. Total DNA was extracted directly from the lyophilized crude venom and the gene (5.5?kbp) coding for Da-36 had been successfully cloned. Sequence analysis revealed that the Da-36 gene contained five exons and four introns. The mature Da-36 was encoded by four separate exons. The deduced mature amino acid sequence of Da-36 was in good agreement with the determined N-terminal sequence of the purified protein and shared high homology with other serine proteases isolated from different snake venoms. Blast search using amino acid sequence of Da-36 against public database revealed that Da-36 showed a maximal identity of 90 % with both Dav-X (Swiss-Prot: ) and thrombin-like protein 1 (GenBank: ) from the same snake species, indicating that Da-36 is a novel serine protease.