Biotechnology letters · 2017
Cloning, expression, purification and bioactivity evaluation of a thrombin-like enzyme from Deinagkistrodon acutus venom gland library.
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Li An, Zhang Chuan, Wang Jie, Wang Junjie, Jiang Hailong, Li Jianzhong, Ma Xingyuan, Zhang Wen, Lu Yiming
Аннотация
OBJECTIVES: To identify a new member of serine proteases from Deinagkistrodon acutus via phage display technique and appraise its biocatalytic activities.
RESULTS: 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.
CONCLUSIONS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
Предметные рубрики MeSH
AnimalsBlood CoagulationCloning, MolecularCrotalinaeElectrophoresis, Polyacrylamide GelEscherichia coliExocrine GlandsFibrinogenGene ExpressionGene LibraryGenetic TestingHumansHydrogen-Ion ConcentrationMolecular WeightOpen Reading FramesPeptide LibraryPlasmaProteolysisRecombinant Fusion ProteinsSerine ProteasesTemperatureVenoms