A high yield and cost-efficient expression system of human granzymes in mammalian cells

摘要: When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor infected with intracellular pathogens, they release their granule content to eliminate the target and pathogen. Death of host pathogens is triggered by serine proteases, granzymes (Gzms), delivered into cell cytosol pore forming protein perforin (PFN) bacterial prokaryotic membrane disrupting granulysin (GNLY). To investigate molecular mechanisms death mediated Gzms in experimental in-vitro settings, expression purification systems that produce high amounts active enzymes are necessary. Mammalian secreted imply potential correctly folded, fully functional bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection HEK293T human cloned plasmid pHLsec. Gzm from culture supernatant was achieved immobilized nickel affinity chromatography using C-terminal polyhistidine tag provided vector. The insertion an enterokinase site at N-terminus allowed generation protease finally purified cation exchange chromatography. system tested producing levels A, B M should be capable virtually every enzyme body yields.