title: An optimized protocol for expression and purification of murine perforin in insect cells.
abstract: Perforin (PFN) is one of the most important protein effectors of the immune system. It is produced by cytotoxic T lymphocytes and natural killer cells and helps with the clearance of virus-infected and tumor cells. PFN is a pore-forming protein that readily binds to the lipid membranes of target cells, oligomerizes at the cell surface and forms transmembrane pores that allow passage of ions and other larger molecules. Its characterization was hindered in the past by a lack of efficient and reliable expression systems that would result in pure and functional product. In this paper we present optimization of PFN expression in a baculovirus expression system. We optimized several parameters of murine PFN (mPFN) expression and purification and showed that the expressed product is pure and hemolytically active and that it forms pores in the plasma membranes of K562 cells. We could also observe circular pores formed on liposome membranes by cryo-electron microscopy (cryo-EM). Our protocol opens the door for further biochemical and biophysical assessment of PFN properties and interactions with small ligands and lipid membranes.
authors.affiliation: Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia. Electronic address: omar.naneh@ki.si.
authors.forename: Omar
authors.initials: O
authors.lastname: Naneh
authors.affiliation: Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia. Electronic address: polona.bedina@ki.si.
authors.forename: Apolonija Bedina
authors.initials: AB
authors.lastname: Zavec
authors.affiliation: Laboratory for Polymer Chemistry and Technology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia. Electronic address: david.pahovnik@ki.si.
authors.forename: David
authors.initials: D
authors.lastname: Pahovnik
authors.affiliation: Laboratory for Polymer Chemistry and Technology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia. Electronic address: ema.zagar@ki.si.
authors.forename: Ema
authors.initials: E
authors.lastname: Žagar
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK. Electronic address: gilbert@strubi.ox.ac.uk.
authors.forename: Robert J C
authors.initials: RJ
authors.lastname: Gilbert
authors.affiliation: Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia. Electronic address: igor.krizaj@ijs.si.
authors.forename: Igor
authors.initials: I
authors.lastname: Križaj
authors.affiliation: Laboratory for Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia; Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia. Electronic address: gregor.anderluh@ki.si.
authors.forename: Gregor
authors.initials: G
authors.lastname: Anderluh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D002462:Cell Membrane / Q000187:drug effects; D020285:Cryoelectron Microscopy; D006801:Humans; D020014:K562 Cells; D051379:Mice; D052899:Pore Forming Cytotoxic Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D020539:Sequence Analysis, Protein; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins; C483886:perforin, mouse
keywords: Baculovirus expression system; Innate immunity; Insect cells; MACPF/CDC superfamily; Perforin; Pore-forming protein