Li An, Zhang Chuan, Wang Jie, Wang Junjie, Jiang Hailong, Li Jianzhong, Ma Xingyuan, Zhang Wen, Lu Yiming
School of Pharmacy, Second Military Medical University, Shanghai, 200433, China.
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China.
Biotechnol Lett. 2018 Jan;40(1):93-102. doi: 10.1007/s10529-017-2441-z. Epub 2017 Sep 21.
To identify a new member of serine proteases from Deinagkistrodon acutus via phage display technique and appraise its biocatalytic activities.
A novel thrombin-like enzyme gene was cloned by screening the phage display library of D. acutus venom gland. The gene has a 783 bp ORF encoding 260 amino acids. A recombinant enzyme expression vector was constructed and the fused protein was expressed in Escherichia coli. The protein was purified showing a single band of approx. 49.4 kDa after SDS-PAGE. The recombinant enzyme was capable of congealing normal human plasma in vitro with the minimum coagulant dose of 6 µg in 57 s. It exhibited fibrinogenolytic activity by hydrolyzing the Aα-chain of human fibrinogen. It was most active at pH 7.5-8.0 and 35-40 °C with the highest clotting activity of 120 NIH units/mg. It was completely inhibited by PMSF but not by EDTA. Multiple sequence alignments demonstrate that this protein shares high identity with other thrombin-like enzymes from snake venoms.
A novel thrombin-like protein from D. acutus venom was identified, expressed and biologically characterized in vitro. Its fibrinogenolytic properties make the enzyme applicable for biochemical research and drug development on thrombolytic therapy.
通过噬菌体展示技术从尖吻蝮中鉴定一种丝氨酸蛋白酶新成员，并评估其生物催化活性。
通过筛选尖吻蝮毒腺噬菌体展示文库克隆了一个新的类凝血酶基因。该基因有一个783 bp的开放阅读框，编码260个氨基酸。构建了重组酶表达载体，并在大肠杆菌中表达融合蛋白。经SDS-PAGE后纯化的蛋白呈现一条约49.4 kDa的单带。重组酶能够在体外使正常人血浆凝固，最低凝血剂量为6 μg，凝血时间为57 s。它通过水解人纤维蛋白原的Aα链表现出纤维蛋白溶解活性。在pH 7.5 - 8.0和35 - 40 °C时活性最高，最高凝血活性为120 NIH单位/mg。它被苯甲基磺酰氟完全抑制，但不被乙二胺四乙酸抑制。多序列比对表明该蛋白与其他蛇毒类凝血酶具有高度同源性。
从尖吻蝮毒液中鉴定、表达了一种新的类凝血酶蛋白，并对其进行了体外生物学特性研究。其纤维蛋白溶解特性使该酶可应用于生化研究和溶栓治疗的药物开发。
