title: A novel expression and purification system for the production of enzymatic and biologically active human granzyme B.
abstract: The serine protease granzyme B (grB) has previously been shown to induce perforin-independent apoptosis in membrane Hsp70 positive tumor cells in a number of in vitro experimental systems. Ongoing studies that are investigating the in vivo relevance of these findings by assessing the therapeutic potential of grB in a human xenograft tumor mouse model required the development of an expression system for the production of high yields of enzymatic and biologically active human grB. In order to maintain potentially important posttranslational modifications that occur in mammalian cells, human embryonal kidney cells (HEK293) were stably transfected with human grB. The HEK293 host cells were protected from apoptotic cell death by fusing an inactivation site coupled to a (His)(6) tag to the gene sequence of GrB. Inactive grB which was actively released from HEK293 cells by insertion of a Igκ leader sequence was purified on a nickel column utilizing the (His)(6) tag. After enterokinase digestion and heparin affinity chromatography, high yields of enzymatic and biologically active human grB were obtained. The perforin-independent interaction of grB with membrane Hsp70 positive tumor cells appeared to be associated with mammalian glycosylation and mediated by the oligosaccharide moiety of neuraminic acid (NANA).
authors: Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universität München, Ismaninger Str. 22, 81675 Munich, Germany.
authors: Mathias
authors: M
authors: Gehrmann
authors: Brigitte T
authors: BT
authors: Doss
authors: Michaela
authors: M
authors: Wagner
authors: Kirstin A
authors: KA
authors: Zettlitz
authors: Roland E
authors: RE
authors: Kontermann
authors: Gemma
authors: G
authors: Foulds
authors: Alan G
authors: AG
authors: Pockley
authors: Gabriele
authors: G
authors: Multhoff
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D053804:Granzymes / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D057809:HEK293 Cells; D018840:HSP70 Heat-Shock Proteins; D006801:Humans; D051379:Mice; D009438:Neuraminic Acids / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D018840:HSP70 Heat-Shock Proteins; D009438:Neuraminic Acids; D011993:Recombinant Fusion Proteins; D053804:Granzymes