title: A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells.
abstract: When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.
authors.affiliation: Cellular and Molecular Medicine Program, Boston Children's Hospital and Harvard Medical School.
authors.forename: Farokh
authors.initials: F
authors.lastname: Dotiwala
authors.affiliation: Department of Medicine, University of Fribourg.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Fellay
authors.affiliation: Department of Medicine, University of Fribourg.
authors.forename: Luis
authors.initials: L
authors.lastname: Filgueira
authors.affiliation: Centre Médical Universitaire, University of Geneva.
authors.forename: Denis
authors.initials: D
authors.lastname: Martinvalet
authors.affiliation: Cellular and Molecular Medicine Program, Boston Children's Hospital and Harvard Medical School.
authors.forename: Judy
authors.initials: J
authors.lastname: Lieberman
authors.affiliation: Department of Medicine, University of Fribourg; michael.walch@unifr.ch.
authors.forename: Michael
authors.initials: M
authors.lastname: Walch
mesh_terms: D002118:Calcium / Q000737:chemistry; D018929:Cell Culture Techniques / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D004765:Enteropeptidase / Q000737:chemistry; D053804:Granzymes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D057809:HEK293 Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics; D014162:Transfection / Q000379:methods*
chemical_list: D053804:Granzymes; D004765:Enteropeptidase; D002118:Calcium