title: Characterization of a recombinant granzyme B derivative as a "restriction" protease.
abstract: Blood coagulation factor Xa (FXa) and Thrombin are well-known serine proteases often used for processing of recombinant fusion proteins, but because they are purified from bovine blood or other animal sources, there is a risk of pathogenic contaminants in the preparation of the proteases. We report here the characterization of a recombinant serine protease produced in Escherichia coli, which can be used as a specific and efficient alternative to FXa and Thrombin as processing protease. This recombinant protease is derived from human granzyme B (GrB). The protease is found to be very stable in general, and it performs very well in the cleavage of several different fusion proteins tested and was even found superior to processing by FXa in two cases.
authors.affiliation: Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark. charm@boreanpharma.com
authors.forename: Charlotte Harkjaer
authors.initials: CH
authors.lastname: Fynbo
authors.forename: Rikke Høegh
authors.initials: RH
authors.lastname: Lorentsen
authors.forename: Michael
authors.initials: M
authors.lastname: Etzerodt
authors.forename: Hans Christian
authors.initials: HC
authors.lastname: Thøgersen
authors.forename: Thor Las
authors.initials: TL
authors.lastname: Holtet
mesh_terms: D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D053804:Granzymes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases