Expression and characterization of functionally active recombinant perforin produced in insect cells.
A key cytolytic mediator used by killer lymphocytes, perforin (also known as pore-forming protein or cytolysin), has been shown to be capable of undergoing polymerization to form pores in cell membranes and cause osmotic lysis of target cells. Although perforin has been purified from killer lymphocytes and the coding gene has been cloned and sequenced, information concerning the domain structure of the perforin molecule has remained scarce. To overcome the difficulty in obtaining sufficient amounts of perforin and to further assess the functional relevance of the N-terminal portion of the perforin molecule in its lytic activity, we have attempted in the present study to produce recombinant perforins. Three forms of recombinant mouse perforin, a full-length form and two N-terminal truncated forms, have been expressed in insect (Spodoptera frugiperda (Sf9)) cells using recombinant baculovirus. Biochemical and functional characterization showed the purified full-length recombinant perforin to be capable of lysing target cells, inducing Ca2+ influx into target cells, and forming structural pores in target membranes. Significant lytic activities were also detected for the two truncated recombinant perforins lacking, respectively, the N-terminal 21 amino acid residues and 121 amino acid residues. Time course study showed that the latter acted less efficiently than the former. These results suggest the N-terminal portion of the perforin molecule to be an important, but not the only, domain responsible for the lytic function of the perforin molecule.
Liu
C C
CC
Laboratory of Molecular Immunology and Cell Biology, The Rockefeller University, New York 10021, USA.
Persechini
P M
PM
Young
J D
JD
D000818:Animals; D016367:Baculoviridae / Q000276:immunology; D003602:Cytotoxicity, Immunologic / Q000187:drug effects*; D005822:Genetic Vectors / Q000378:metabolism*; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000276:immunology; D008223:Lymphoma / Q000276:immunology; D012515:Mast-Cell Sarcoma / Q000276:immunology; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D051379:Mice; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000494:pharmacology* / Q000648:ultrastructure; D018411:Spodoptera / Q000166:cytology / Q000235:genetics*; D014407:Tumor Cells, Cultured
D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins; D054353:Perforin