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.
Keywords: Baculovirus expression system; Innate immunity; Insect cells; MACPF/CDC superfamily; Perforin; Pore-forming protein.
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