文献类型：期刊文献

英文题名：Cloning, expression, purification and bioactivity evaluation of a thrombin-like enzyme from Deinagkistrodon acutus venom gland library

作者：Li, An[1];Zhang, Chuan[2];Wang, Jie;Wang, Junjie;Jiang, Hailong[1];Li, Jianzhong[1];Ma, Xingyuan[2];Zhang, Wen[1];Lu, Yiming[1]

机构：[1]Second Mil Med Univ, Sch Pharm, Shanghai 200433, Peoples R China;[2]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China; Second Mil Med Univ, Changhai Hosp, Shanghai 200433, Peoples R China

年份：2018

卷号：40

期号：1

起止页码：93

外文期刊名：BIOTECHNOLOGY LETTERS

收录：;EI(收录号：20173904204219);WOS:【SCI-EXPANDED(收录号:WOS:000424228200012)】；

基金：This work was supported by the National Natural Science Foundation of China (No. 30500093 and No. 81274162), Shanghai Science and Technology Innovation Action Plan (No. 16431904400), Innovation Program of Shanghai Municipal Education Commission (No. 14ZZ077), National Major Scientific and Technological Special Project for "Significant New Drugs Development" (No. 2009ZX09103-690) and Army Medical Hygiene Research Foundation (No. 06Q042).

语种：英文

外文关键词：Deinagkistrodon acutus; Blood clotting; Fibrinogenolytic activity; Phage display; Snake venom; Thrombin-like enzyme

摘要：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 A mu g in 57 s. It exhibited fibrinogenolytic activity by hydrolyzing the A alpha-chain of human fibrinogen. It was most active at pH 7.5-8.0 and 35-40 A degrees 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.