title: Expression and characterization of recombinant ecarin.
abstract: The snake venom protease ecarin from Echis carinatus was expressed in stable transfected CHO-S cells grown in animal component free cell culture medium. Recombinant ecarin (r-ecarin) was secreted from the suspension adapted Chinese Hamster Ovary (CHO-S) host cells as a pro-protein and activation to the mature form of r-ecarin occurred spontaneously during continued incubation of the cell culture at 37 °C after death of the host cells. Maximal ecarin activity was reached 7 days or more after cell culture viability had dropped to zero. The best producing CHO-S clone obtained produced up to 7,000 EU ecarin/litre in lab scale shaker cultures. The conversion of different concentrations of both prothrombin and prethrombin-2 as substrates for native and r-ecarin were examined with a chromogenic thrombin substrate. At low concentrations both these proteins were converted into thrombin by the two ecarin preparations with comparable rates. However, with prothrombin concentrations above 250 nM r-ecarin apparently had a two times higher turnover than native ecarin, consistent with the observed rapid complete conversion of prothrombin into thrombin by r-ecarin. With r-ecarin a K (m) value of 0.4 μM prethrombin-2 was determined but only a rough estimate could be made of the K (m) for prothrombin of 0.9 μM. In conclusion, r-ecarin was identified as a promising candidate for replacement of native ecarin in assays utilizing conversion of prothrombin to thrombin.
authors.affiliation: AstraZeneca R&D, 43183, Mölndal, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Jonebring
authors.forename: Ute
authors.initials: U
authors.lastname: Lange
authors.forename: Elke
authors.initials: E
authors.lastname: Bucha
authors.forename: Johanna
authors.initials: J
authors.lastname: Deinum
authors.forename: Margareta
authors.initials: M
authors.lastname: Elg
authors.forename: Ann
authors.initials: A
authors.lastname: Lövgren
mesh_terms: D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D005343:Fibrinolytic Agents / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D011516:Prothrombin / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014757:Viper Venoms / Q000235:genetics* / Q000378:metabolism; D017819:Viperidae / Q000235:genetics / Q000378:metabolism*
chemical_list: D005343:Fibrinolytic Agents; D011994:Recombinant Proteins; D014757:Viper Venoms; D011516:Prothrombin; D010450:Endopeptidases; C020097:ecarin