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Expression and characterization of functionally active recombinant perforin produced in insect cells
C C Liu1, P M Persechini, J D Young
1Laboratory of Molecular Immunology and Cell Biology, The Rockefeller University, New York 10021, USA.
Journal of Immunology (Baltimore, Md. : 1950)
|May 1, 1996
Summary
Recombinant perforins were produced to study their pore-forming ability. The N-terminal region is crucial for perforin
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Perforin is a key cytolytic mediator produced by killer lymphocytes.
- It forms pores in cell membranes, leading to target cell lysis.
- Limited information exists on perforin's domain structure and its role in lytic activity.
Purpose of the Study:
- To produce recombinant mouse perforins to investigate their structure-function relationship.
- To assess the functional relevance of the N-terminal portion of perforin in its lytic activity.
Main Methods:
- Expression of full-length and N-terminally truncated recombinant mouse perforins in insect cells (Sf9) using baculovirus.
- Biochemical and functional characterization of recombinant perforins, including cell lysis assays, Ca2+ influx measurements, and pore formation studies.
Main Results:
- Purified full-length recombinant perforin demonstrated cell-lysing activity, induced Ca2+ influx, and formed pores in target membranes.
- Truncated perforins lacking 21 or 121 N-terminal amino acids retained significant lytic activity.
- The perforin lacking 121 amino acids showed reduced lytic efficiency compared to the one lacking 21 amino acids.
Conclusions:
- The N-terminal portion of the perforin molecule is important for its lytic function.
- However, other domains also contribute to perforin's pore-forming and cell-lysing capabilities.