EXPRESSION AND CHARACTERIZATION OF FUNCTIONALLY ACTIVE RECOMBINANT PERFORIN PRODUCED IN INSECT CELLS

A key cytolytic mediator used by killer lymphocytes, perforin (also kn own as pore-forming protein or cytolysin), has been shown to be capabl e of undergoing polymerization to form pores in cell membranes and cau se osmotic lysis of target cells, Although perforin has been purified from killer lymphocytes and the coding gene has been cloned and sequen ced, information concerning the domain structure of the perforin molec ule has remained scarce, To overcome the difficulty in obtaining suffi cient amounts of perforin and to further assess the functional relevan ce of the N-terminal portion of the perforin molecule in its lytic act ivity, we have attempted in the present study to produce recombinant p erforins. Three forms of recombinant mouse perforin, a full-length for m and two N-terminal truncated forms, have been expressed in insect (S podoptera frugiperda (Sf9)) cells using recombinant baculovirus. Bioch emical and functional characterization showed the purified full-length recombinant perforin to be capable of lysing target cells, inducing C a2+ influx into target cells, and forming structural pores in target m embranes, 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 r esults suggest the N-terminal portion of the perforin molecule to be a n important, but not the only, domain responsible for the lytic functi on of the perforin molecule.