title: Expression, refolding, and purification of recombinant human granzyme B.
abstract: Granzyme B (GrB) is a member of a family of serine proteases involved in cytotoxic T-lymphocyte-mediated killing of potentially harmful cells, where GrB induces apoptosis by cleavage of a limited number of substrates. To investigate the suitability of GrB as an enzyme for specific fusion protein cleavage, two derivatives of human GrB, one dependent on blood coagulation factor Xa (FXa) cleavage for activation and one engineered to be self-activating, were recombinantly expressed in Escherichia coli. Both derivatives contain a hexa-histidine affinity tag fused to the C-terminus and expressed as inclusion bodies. These were isolated and solubilized in guanidiniumHCl, immobilized on a Ni2+-NTA agarose column, and refolded by application of a cyclic refolding protocol. The refolded pro-rGrB-H6 could be converted to a fully active form by cleavage with FXa or, for pro(IEPD)-rGrB-H6, by autocatalytic processing during the final purification step. A self-activating derivative in which the unpaired cysteine of human GrB was substituted with phenylalanine was also prepared. Both rGrB-H6 and the C228F mutant were found to be highly specific and efficient processing enzymes for the cleavage of fusion proteins, as demonstrated by cleavage of fusion proteins containing the IEPD recognition sequence of GrB.
authors.affiliation: Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark. rhl@boreanpharma.com
authors.forename: Rikke H
authors.initials: RH
authors.lastname: Lorentsen
authors.forename: Charlotte H
authors.initials: CH
authors.lastname: Fynbo
authors.forename: Hans C
authors.initials: HC
authors.lastname: Thøgersen
authors.forename: Michael
authors.initials: M
authors.lastname: Etzerodt
authors.forename: Thor L
authors.initials: TL
authors.lastname: Holtet
mesh_terms: D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D053804:Granzymes; D006801:Humans; D007700:Kinetics; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011993:Recombinant Fusion Proteins; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases